Potentiation of the anti-tumour effect of docetaxel by conjugated linoleic acids (CLAs) in breast cancer cells in vitro

Response rates of tumours to docetaxel (DOCT) are 45-60% in advanced breast cancer but problems associated with side effects, drug resistance and high costs occur. Conjugated linoleic acids (CLAs) also have anti-tumorigenic activity that elicits similar changes in oncogene expression to DOCT and could augment DOCT efficacy. CLA isomers appear to differ in cytotoxicity toward cancer cells. Effects of two CLA isomers on cytotoxicity of DOCT in breast cancer cells (MCF-7; MDA-MB-231) in vitro were assessed. Cells were incubated up to 72 h with 40 microM each of LA or CLA isomers (cis-9, trans-10 CLA, or trans-10, cis-12 CLA) or a 50:50 isomer mix, alone or with DOCT (0-64 microM); a pilot study determined IC(50) and IC(70) concentrations. Treatments were concurrent (CLA and DOCT together) or sequential (CLA then DOCT). MTT assay determined cell viability. Trans-10, cis-12 CLA was the most effective fatty acid (P<0.001) and increased with treatment time. IC(50) and IC(70) concentrations of DOCT were determined, concurrently or sequentially, with and without fatty acids, in the two cell types. Concurrent treatment with trans-10, cis-12 CLA and DOCT augmented inhibition of cell growth in one or both cell lines (decreased IC(50) and IC(70) in MCF-7; P<0.05 but only IC(50) in MDA-MB-231; P<0.05). CLA mix reduced IC(50) and IC(70) in MDA-MB-231 (P<0.001) but not in MCF-7. Cis-9, trans-11 CLA and LA had no effect. Sequential treatment with CLAs then DOCT reduced IC(50) and IC(70) in MCF-7 but not in MDA-MB-231. The latter had increased IC(50) and IC(70) with LA treatment (P<0.05) and increased IC(70) with cis-9, trans-11 CLA (P<0.05) with sequential but not concurrent treatment. Longer pre-incubation times with trans-10, cis-12 CLA (24-72 h) elicited greater reductions in IC(50) and IC(70) in MCF-7 cells. Results show that CLA isomers augment anti-tumour effects of docetaxel in breast cancer cells and suggest possible dual treatment regimens.     

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells

BackgroundBreast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.Hypothesis/purposeThe present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellata in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.Study designThe inhibitory effects of Saxi A on cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.MethodsMTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.ResultsOur results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shERα-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ERα remained viable.ConclusionSaxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ERα status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER⁺) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER⁺ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER⁺ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66^?/PR^?/HER2^?) and ER-α36⁺/GPER1⁺ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER⁺ breast cancer.     

Indoleamine 2,3-dioxygenase activity and l-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with l-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-γ (1000 units/ml). Accordingly, l-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-γ. 1-Methyl-dl-tryptophan (1 mM) inhibited interferon-γ induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-γ. l-Tryptophan transport into MDA-MB-231 cells was via a Na⁺-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-d,l-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, l-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.     

Actaea racemosa L. extract inhibits steroid sulfation in human breast cancer cells: Effects on androgen formation

BackgroundActaea racemosa L., also known as black cohosh, is a popular herb commonly used for the treatment of menopausal symptoms. Because of its purported estrogenic activity, black cohosh root extract (BCE) may trigger breast cancer growth.Study design/methodsThe potential effects of standardized BCE and its main constituent actein on cellular growth rates and steroid hormone metabolism were investigated in estrogen receptor alpha positive (ERα+) MCF-7 and -negative (ERα-) MDA-MB-231 human breast cancer cells. Cell numbers were determined following incubation of both cell lines with the steroid hormone precursors dehydroepiandrosterone (DHEA) and estrone (E1) for 48 h, in the presence and absence of BCE or actein. Using a validated liquid chromatography-high resolution mass spectrometry assay, cell culture supernatants were simultaneously analyzed for the ten main steroids of the estrogen pathway.ResultsInhibition of MCF-7 and MDA-MB-231 cell growth (up to 36.9%) was observed following treatment with BCE (1-25 µg/ml) or actein (1-50 µM). Incubation of MCF-7, but not of MDA-MB-231 cells, with DHEA and BCE caused a 20.9% reduction in DHEA-3-O-sulfate (DHEA-S) formation, leading to a concomitant increase in the androgens 4-androstene-3,17-dione (AD) and testosterone (T). Actein was shown to exert an even stronger inhibitory effect on DHEA-S formation in MCF-7 cells (up to 89.6%) and consequently resulted in 12- to 15-fold higher androgen levels compared with BCE. The formation of 17β-estradiol (E2) and its glucuronidated and sulfated metabolites was not affected by BCE or actein after incubation with the estrogen precursor estrone (E1) in either cell line.ConclusionsThe results of the present study demonstrated that actein and BCE do not promote breast cancer cell growth or influence estrogen levels. However, androgen formation was strongly stimulated by BCE and actein, which may contribute to their ameliorating effects on menopausal symptoms in women. Future studies monitoring the levels of AD and T upon BCE supplementation of patients are warranted to verify an association between BCE and endogenous androgen metabolism.     

Effect of fetal bovine serum on erythropoietin receptor expression and viability of breast cancer cells

Erythropoietin receptors (EPORs) are present not only in erythrocyte precursors but also in non-hematopoietic cells including cancer cells. In this study, we determined the effect of fetal bovine serum (FBS) in culture medium on the EPOR expression and viability of the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Using flow cytometry, we showed that the inclusion of 10% FBS in the medium increased the EPOR expressions and viabilities of MDA-MB-231 and MCF-7 cells. The MDA-MB-231 showed greater EPOR expression than MCF-7 cells, suggesting that the presence of ERs on cells is associated with poor expression of EPOR. Culture medium containing 10% FBS also caused increased number of breast cancer cells entering the synthesis phase of the cell cycle. The study also showed that rHuEPO treatment did not affect viability of breast cancer cells. In conclusion, it was shown that the inclusion of FBS in culture medium increased expression of EPOR in breast cancer cells and rHuEPO treatment had no effect on the proliferation of these cancer cells.     

Synthesis and anti breast cancer activity of biphenyl based chalcones

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33) were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer) cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 μM. The two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell cycle arrest and loss of mitochondrial membrane potential.     

Effects of triptolide from Tripterygium wilfordii on ERα and p53 expression in two human breast cancer cell lines

The aim of the study was to discover possible differential cytotoxicity of triptolide towards estrogen-sensitive MCF-7 versus estrogen-insensitive MDA-MB-231 human breast cancer cells. Considering that MCF-7 cells express functional Estrogen receptor α (ERα) and wild-type p53, whereas MDA-MB-231 cells which are ERα-negative express mutant p53, the anti-proliferation effect of triptolide on MCF-7 and MDA-MB-231 cells were examined, the apoptotic effect and cell cycle arrest caused by triptolide were investigated, ERα and p53 expression were also observed in this paper. The results showed that the anti-proliferation effects were induced by triptolide in both cell lines. But the value of IC₅₀ in MCF-7 cells for its anti-proliferation effect was about one tenth of that in MDA-MB-231 cells, which indicated that the effect is more potent in MCF-7 cells. Condensed chromatin or fragmented nuclei could be found in MCF-7 cells treated with only 40 nM triptolide but in MDA-MB-231 cells they couldn’t be observed until the concentration reached to 400 nM. Triptolide induced significant S cell cycle arrest along with the presence of sub-G0/G1 peak in MDA-MB-231 cells, whereas there was only slightly S cell cycle arrest on cell cycle distribution in MCF-7 cells. The role of p53 in two breast cancer cells was examined, the results showed that the mutant p53 in MDA-MB-231 cells was suppressed and the wild-type p53 in MCF-7 was increased. Moreover, triptolide could down regulate the expression of ERα in MCF-7 cells. The results showed that triptolide is much more sensitive to ERα-positive MCF-7 cells than to ERα-negative MDA-MB-231 cells, and the sensitivity is significantly associated with the ERα and p53 status.     

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells

BackgroundCoumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed.MethodsAntiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot.ResultsThe inhibition concentration (IC₅₀) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC₅₀) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84 μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process.ConclusionStyrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.