Agent For Treatment Of Breast Cancer Research Articles

PCP4/PEP19 promotes migration, invasion and adhesion in human breast cancer MCF-7 and T47D cells.

Purkinje cell protein (PCP) 4/peptide (PEP) 19 is expressed in Purkinje cells where it has a calmodulin-binding, anti-apoptotic function. We recently demonstrated that PCP4/PEP19 is expressed and inhibit apoptosis in human breast cancer cell lines. In the present study we investigated the role of PCP4/PEP19 in cell morphology, adhesion, migration, and invasion in MCF-7 and T47D human breast cancer cell lines. Knockdown of PCP4/PEP19 reduced the formation of filopodia-like cytoplasmic structures and vinculin expression, and enhanced E-cadherin expression. Activities of migration, invasion, and cell adhesion were also decreased after the knockdown of PCP4/PEP19 in MCF-7 and T47D cells. These results suggested that PCP4/PEP19 promotes cancer cell adhesion, migration, and invasion and that PCP4/PEP19 may be a potential target for therapeutic agents in breast cancer treatment which act by inhibiting epithelial-mesenchymal transition and enhancing apoptotic cell death.

Cordycepin, a Natural Antineoplastic Agent, Induces Apoptosis of Breast Cancer Cells via Caspase-dependent Pathways

Cordycepin, a major compound separated from Cordyceps sinensis, is known as a potential novel candidate for cancer therapy. Breast cancer, the most typical cancer diagnosed among women, remains a global health problem. In this study, the anti-breast cancer property of cordycepin and its underlying mechanisms was investigated. The direct effects of cordycepin on breast cancer cells both in in vitro and in vivo experiments were evaluated. Cordycepin exerted cytotoxicity in MCF-7 and MDA-MB-231 cells confirmed by reduced cell viability, inhibition of cell proliferation, enhanced lactate dehydrogenase release and reactive oxygen species accumulation, induced mitochondrial dysfunction and nuclear apoptosis in human breast cancer cells. Cordycepin increased the activation of pro-apoptotic proteins, including caspase-8, caspase-9, caspase-3 and Bax, and suppressed the expression of the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2). The inhibition on MCF-7-xenografted tumor growth in nude mice further confirmed cordycepin's anti-breast cancer effect. These aforementioned results reveal that cordycepin induces apoptosis in human breast cancer cells via caspase-dependent pathways. The data shed light on the possibility of cordycepin being a safe agent for breast cancer treatment.

Inhibitory Effect of Ginseng on Breast Cancer Cell Line Growth Via Up-Regulation of Cyclin Dependent Kinase Inhibitor, p21 and p53

Objective:Breast cancer is global female health problem worldwide. Most of the currently used agents for breast cancer treatment have toxic side-effects. Ginseng root, an oriental medicine, has many health benefits and may exhibit direct anti-cancer properties. This study was performed to assess the effects of ginseng on breast cancer cell lines.Materials and Methods:Cytotoxicity of ginseng extract was measured by MTT assay after exposure of MDA-MB-231, MCF-10A and MCF-7 breast cancer cells to concentrations of 0.25, 0.5, 1, 1.5, 2 and 2.5 mg/well. Expression levels of p21WAF, p16INK4A, Bcl-2, Bax and P53 genes were analyzed by quantitative real time PCR.Results:The treatment resulted in inhibition of cell proliferation in a dose-and time-dependent manner. p53, p21WAF1and p16INK4A expression levels were up-regulated in ginseng treated MDA-MB-231 and MCF-7 cancer cells compared to untreated controls and in MCF-10A cells. The expression levels of Bcl2 in the MDA-MB-231 and MCF-7 cells were down-regulated. In contrast, that of Bax was significantly up-regulated.Conclusion:The results of this study revealed that ginseng may inhibit breast cancer cell growth by activation of the apoptotic pathway.

Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors That Also Modulate Estrogen Receptors.

A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor α (ER-α) and estrogen receptor β (ER-β), and antagonize the activity of β-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and high ER-α and ER-β binding affinities of several of the resulting analogues, together with the facts that they antagonize β-estradiol in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in order to obtain dual AI/SERM agents for breast cancer treatment.

MiR-129-3p promotes docetaxel resistance of breast cancer cells via CP110 inhibition

Docetaxel is commonly used as an effective chemotherapeutic agent in breast cancer treatment, but the underlying mechanisms of drug resistance are not fully understood. The purpose of this study was to investigate the possible role of miR-129-3p in breast cancer cell resistance to docetaxel. MiR-129 and miR-129-3p inhibitor were transfected into breast cancer cells to investigate their effects on chemoresistance to docetaxel. The function of miR-129-3p was evaluated by apoptosis, cell proliferation, and cell cycle assays. We found that miR-129-3p was up-regulated in MDA-MB-231/Doc cells, concurrent with CP110 down-regulation, compared to the parental MDA-MB-231 cells. In vitro drug sensitivity assays demonstrated that miR-129-3p inhibition sensitized MDA-MB-231/Doc and MCF-7 cells to docetaxel, whereas miR-129 overexpression enhanced MDA-MB-231 and MCF-7 cell resistance to docetaxel. Ectopic miR-129 expression reduced CP110 expression and the luciferase activity of a CP110 3′ untranslated region-based reporter construct in MDA-MB-231 cells, suggesting that CP110 is a direct miR-129-3p target. We demonstrated that restoration of CP110 expression in MDA-MB-231 and MCF-7 cells by miR-129 overexpression rendered the cells sensitive to docetaxel. In a nude xenograft model, miR-129 up-regulation significantly decreased MDA-MB-231 cells’ response to docetaxel. Our findings suggest that miR-129-3p down-regulation potentially sensitizes breast cancer cells to docetaxel treatment.

In vitro effects of tamoxifen on adipose-derived stem cells.

In breast reconstructive procedures, adipose-derived stem cells (ASCs) that are present in clinical fat grafting isolates are considered to play the main role in improving wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. However, it is unclear if tamoxifen (TAM) as the most widely used hormonal therapeutic agent for breast cancer treatment, affects the ASCs and ultimately wound healing. This study evaluated whether TAM exposure to in vitro human ASCs modulate cellular functions. Human ASCs were isolated and treated with TAM at various concentrations. The effects of TAM on cell cycle, cell viability and proliferation rates of ASCs were examined by growth curves, MTT assay and BrdU incorporation, respectively. Annexin V and JC-1 Mitochondrial Membrane Potential assays were used to analyze ASC apoptosis rates. ASCs were cultured in derivative-specific differentiation media with or without TAM (5 uM) for 3 weeks. Adipogenic and osteogenic differentiation levels were measured by quantitative RT-PCR and histological staining. TAM has cytotoxic effects on human ASCs through apoptosis and inhibition of proliferation in dose- and time-dependent manners. TAM treatment significantly down-regulates the capacity of ASCs for adipogenic and osteogenic differentiation (p<0.05 vs. control), and inhibit the ability of the ASCs to subsequently formed cords in Matrigel. This study is the first findings to our knowledge that demonstrated that TAM inhibited ASC proliferation and multi-lineage ASC differentiation rates. These results may provide insight into the role of TAM with associated poor soft tissue wound healing and decreased fat graft survival in cancer patients receiving TAM.

Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells

Context: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments.Objective: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells.Materials and methods: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation.Results: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h.Discussion and conclusion: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.

Abstract P3-05-25: Efficacy of everolimus on multiple mechanisms of AI-resistance in vitro and xenograft, and characterization of their everolimus-resistance

Abstract Background: mTOR inhibitor, everolimus showed remarkable clinical efficacy and has been considered as a promising agent for breast cancer treatment especially among hormone receptor positive (HR+) postmenopausal advanced breast cancer. Now Affinitor (everolimus) is approved for HR+ breast cancer in Japan. However, definitive biomarker has been unclear so far. In addition, the subsequent treatment after refractory to everolimus has not been defined. In preclinical study, it has been reported there are several resistant models of endocrine therapy and we also reported several types of models established from MCF-7-E10, in which estrogen receptor (ER) activity could be monitored by transfected-ERE-GFP. Those EDR cells are characterized as the followings: 1) Type 1 shows constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 shows low expression of ER (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated (Fujiki, 2014, J Steroid Biochem Mol Biol). 3) Type4, which was cultured under the condition of estrogen depletion and addition of testosterone. It showed using androgen metabolite as ligand and both PI3K/Akt/mTOR and MAPK signaling pathway upregulated (Hanamura, 2013, Breast Cancer Res Tr). Methods: Efficacy of everolimus was analyzed in those cell lines in vitro and in vivo about inhibition of cell proliferation. In vivo study, ovarectomized mice were inoculated with Type1 or Type4. Treatments were performed as single agent letrozole, everolimus, or combination and tumor volume changes were compared. Then everolimus resistant cells were generated from those EDR cells after long term exposure to everolimus in vitro. Though we could not acquire everolimus resistant cell from Type4, Type1 and 2 showed its resistance. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy between AI and everolimus after the first line AI treatment failure. Using those cell lines, we investigated the different mechanism of resistance to everolimus. Results: Everolimus extremely inhibited cell proliferation in each EDR type in vitro and in vivo. Besides, Type4 was more remarkably sensitive than parental MCF-7-E10 under testosterone supplemented condition in vitro. Everolimus or combination treatments reached significant tumor volume reduction around 50 percent after 21 days treatment period, in contrast to increase of tumor volume in placebo or letrozole group. In vitro study of developing resistance to everolimus, Type1-EVR cells no longer responded to everolimus and surprisingly fulvestrant also lost sensitivity to EVR cell in spite of keeping ERE activity. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than parental cell, however, MEK inhibitor or PI3K inhibitor alone was not effective but combination with fulvestrant significantly effective more than parental cell. In type2, JNK inhibitor was effective for parental cell but EVR cell lost sensitivity to JNK inhibitor, however, Src inhibitor was much more effective in Type2-EVR than in parental cell. Conclusion: Everolimus showed remarkable efficacy to any types of EDR cells, however, the mechanism of acquired resistance to everolimus was likely to be different between each EVR cell. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance. Citation Format: Mariko Kimura, Natsu Fujiki, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Takashi Ishikawa, Itaru Endo, Shin-ichi Hayashi. Efficacy of everolimus on multiple mechanisms of AI-resistance in vitro and xenograft, and characterization of their everolimus-resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-25.

Cytotoxicity of Vitex trifolia leaf extracts on MCF-7 and Vero cell lines

Pharmacological and preventive properties of Vitex trifolia leaf extracts are well known, but the anticancer activity of methanol and petroleum ether extracts of Vitex trifolia leafs on human breast cancer cells have not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against MCF-7 and Vero cell line. Cells were exposed to 125 to 500 μg/ml of the extracts of Vitex trifolia for 72 h. Post-treatment, percent cell viability was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-biphenyl tetrazolium bromide (MTT) assays. The results showed that petroleum ether and methanol extracts significantly reduced cell viability of MCF-7 cells in a concentration dependent manner. Concentrations of 125 μg/ml and above of petroleum ether and 500 μg/ml of methanol extract were found to be cytotoxic in MCF-7 cells. Cell mortality at 125, 250 and 500 μg/ml of petroleum ether extract was recorded as 98.80%, 93.40% and 87.75% respectively, whereas at 125, 250 and 500 of methanol extract values were 79.98%, 75.70% and 70.25%, respectively by MTT assay. MCF-7 and Vero cells exposed to 125, 250 and 500 μg/ml. The data revealed that the treatment with petroleum ether and methanol of Vitex trifolia leaf extract induced cell death in MCF-7 cells. Meanwhile the same extract showed a moderate inhibition against Vero cell lines. It may be concluded that Vitex trifolia can cause cell death in MCF-7 cancer cells, which can be considered as a promising chemotherapeutic agent in breast cancer treatment.

The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells.

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and β-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

MiR-193b Modulates Resistance to Doxorubicin in Human Breast Cancer Cells by Downregulating MCL-1.

MicroRNAs (miRNAs) family, which is involved in cancer development, proliferation, apoptosis, and drug resistance, is a group of noncoding RNAs that modulate the expression of oncogenes and antioncogenes. Doxorubicin is an active cytotoxic agent for breast cancer treatment, but the acquisition of doxorubicin resistance is a common and critical limitation to cancer therapy. The aim of this study was to investigate whether miR-193b mediated the resistance of breast cancer cells to doxorubicin by targeting myeloid cell leukemia-1 (MCL-1). In this study, we found that miR-193b levels were significantly lower in doxorubicin-resistant MCF-7 (MCF-7/DOXR) cells than in the parental MCF-7 cells. We observed that exogenous miR-193b significantly suppressed the ability of MCF-7/DOXR cells to resist doxorubicin. It demonstrated that miR-193b directly targeted MCL-1 3′-UTR (3′-Untranslated Regions). Further studies indicated that miR-193b sensitized MCF-7/DOXR cells to doxorubicin through a mechanism involving the downregulation of MCL-1. Together, our findings provide evidence that the modulation of miR-193b may represent a novel therapeutic target for the treatment of breast cancer.

SRT1720 induces lysosomal-dependent cell death of breast cancer cells.

SRT1720 is an activator of SIRT1, a NAD(+)-dependent protein and histone deacetylase that plays an important role in numerous biologic processes. Several studies have illustrated that SRT1720 treatment could improve metabolic conditions in mouse models and in a study in cancer SRT1720 caused increased apoptosis of myeloma cells. However, the effect of SRT1720 on cancer may be complex, as some recent studies have demonstrated that SRT1720 may not directly activate SIRT1 and another study showed that SRT1720 treatment could promote lung metastasis. To further investigate the role of SRT1720 in breast cancer, we treated SIRT1 knockdown and control breast cancer cell lines with SRT1720 both in vitro and in vivo. We showed that SRT1720 more effectively decreased the viability of basal-type MDA-MB-231 and BT20 cells as compared with luminal-type MCF-7 breast cancer cells or nontumorigenic MCF-10A cells. We demonstrated that SRT1720 induced lysosomal membrane permeabilization and necrosis, which could be blocked by lysosomal inhibitors. In contrast, SRT1720-induced cell death occurred in vitro irrespective of SIRT1 status, whereas in nude mice, SRT1720 exhibited a more profound effect in inhibiting the growth of allograft tumors of SIRT1 proficient cells as compared with tumors of SIRT1-deficient cells. Thus, SRT1720 causes lysosomal-dependent necrosis and may be used as a therapeutic agent for breast cancer treatment.

Abstract 2110: Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7

Abstract mTOR inhibitor everolimus has been considered as a promising agent for breast cancer treatment especially among estrogen receptor (ER) positive postmenopausal advanced breast cancer. As expected, combination therapy of everolimus and steroidal aromatase inhibitor (AI) showed remarkable clinical efficacy for the first line nonsteroidal AI treatment failure patients in BOLERO-2 trial. However, the subsequent treatment after refractory to that therapy remains a great matter. We already reported several types of long term estrogen-depletion resistant (EDR) cell lines from MCF-7-E10, which has been constitutively ERE-GFP-transfected to monitor ER activity in living cells (Fujiki N et al., J Steroid Biochem Mol Biol. 2013 Oct 13;139C:61-72). They mimic AI resistance and these EDR cells have been characterized as the followings. 1) Type 1 as ER positive (ERE-GFP positive), constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 as ER negative conversion (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated. Then with those two types of sub-cell lines, we generated everolimus resistant cells after long term exposure to everolimus in estrogen depleted medium. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy of AI and everolimus as the second line endocrine treatment after the first line AI treatment failure. Though everolimus extremely inhibited cell proliferation of two EDR cell lines, everolimus resistant cells named as Type 1-EVR, Type 2-EVR no longer responded to everolimus. ER expression and estrogen sensitivity of two types of EVR cells were similar to those of the parental cells, respectively. Pure anti-estrogen fulvestrant inhibited cell proliferation of Type1-EVR cells. On the other hand, Type2-EVR did not respond to fulvestrant but JNK inhibitor inhibited the cell growth. Phosphorylation status of p70S6K and 4E-BP1 were different among individual parental EDR cells and EVR cells. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than the parental cells. In contrast, Type2-EVR expressed phosphorylation of MAP-kinase less than the parental cells. As above, the mechanism of acquired resistance to everolimus was likely to be different between those two sub-cell lines. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance. Citation Format: Mariko Kimura, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Itaru Endo, Shin-ichi Hayashi. Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2014-2110

Cytotoxicity Screening of Plants of Genus &lt;i&gt;Piper&lt;/i&gt; in Breast Cancer Cell Lines

Purpose: To examine whether seven species of plants of genus Piper possess anti-cancer effects.Methods: One normal breast and three breast cancer cell lines were used to test cytotoxic effects over a period of 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The dried plants were extracted with methanol and dichloromethane, and the effective extract isolated by crystallization, acid/base extraction and column chromatography techniques. Fragmented DNA was purified by phenol/chloroform/isoamyl alcohol.Results: Methanol and dichloromethane extracts of Piper retrofractum, Piper betle, especially Piper nigrum, exhibited strong effect on MDA-MB-468. When the crude extract of P. nigrum was then separated by column chromatography, fraction D showed activity against both MCF-7 and MDA-MB-468 cells. Fraction DE that was isolated from D demonstrated a highly cytotoxic effect with IC50 values of 8.33 ± 1.27 and 7.48 ± 0.57 μg/ml on MCF-7 and MDA-MB-468 cells, respectively. Furthermore, fraction DF exhibited a strong cytotoxic effect only on MCF-7 with IC50 value of 6.51 ± 0.39 μg/ml. DNA smears of MCF-7 and MDA-MB-468 cells treated with fraction DE and DF were observed within 7 days.Conclusions: These results indicate that the compounds isolated from P. nigrum, viz, DE and DF, have cytotoxic effect on breast cancer cell lines. These fractions could be promising agent for breast cancer treatment. Further studies on the isolation, structural and mechanism elucidation of the active compound are still needed being carried out.Keywords: Cytotoxicity, Breast cancer, P. nigrum, DNA fragmentation

Synergistic anti-carcinogenic effect of interferon-β with cisplatin on human breast adenocarcinoma MDA MB231 cells.

Cisplatin is one of the most commonly used chemotherapeutic agents for breast cancer treatment. However, its efficacy is greatly limited by its toxic side effects. The present study investigated the synergistic effect of interferon β with cisplatin on MDA MB231 cells. The antiproliferative effect was measured by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) was calculated using the method of Chou and Talalay. Cytotoxicity was determined by trypan blue and clonogenic assay. Genotoxic and cytostatic effects were studied using micronucleus assay and nuclear division index (NDI). Protein expression was analyzed using immunoblotting. Interferon β (100-2500 IU/mL) and Cisplatin (0.01-100 μM) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 1500 IU/mL and 20 μM for interferon β and cisplatin, respectively. Western blot analysis revealed expression of interferon β binding receptor in MDA MB231 cells. More interestingly, synergistic, cytotoxic and genotoxic effects were observed after treatment with a combination of interferon β with reduced dosage of cisplatin. Decreased expression of Bcl-2 and increased expression of Bax stimulated the cytochrome c release, which triggers caspase-9 and -3 activation significantly increased in the combinational group. In conclusion the combination of interferon β with reduced dose of cisplatin results synergistically improved growth-inhibition and apoptosis-inducing effect on MDA MB231 cells.

Interplay between estrogen and retinoid signaling in breast cancer – Current and future perspectives

All-trans-retinoic acid (RA) is a promising agent for breast cancer treatment, but it induces several adverse effects and the few clinical trials performed up to now in breast cancer patients have provided disappointing results. The combination of RA and antiestrogenic compounds, such as tamoxifen, synergistically decreases the proliferation of breast cancer cells and an interplay between retinoid and estrogen signaling has begun to be unraveled, turning these combinations into an appealing strategy for breast cancer treatment. This review focus on the current knowledge regarding the interplay between retinoid and estrogen signaling in breast cancer and the combinations of RA with antiestrogens, aiming their future utilization in cancer therapy.

The pro-apoptotic role of autophagy in breast cancer.

Background:Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines.Methods:Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis.Results:LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells.Conclusions:Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa.

Identifying molecular features that distinguish fluvastatin-sensitive breast tumor cells

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatintriggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERα)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genesencoding regulators of mevalonate production, non-sterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug, Gemcitabine

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking or reversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategies to reduce cancer related death. One such promising chemopreventive agent which has attracted considerable attention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. The present study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent, gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the results by nuclear morphological examination. Further we analyzed the modulation of expression of Bcl-2 and COX-2 on treatment of these cells with SFN by RT-PCR. SFN showed cytotoxic effects on MCF-7 cells in a dose- and time-dependent manner via an apoptotic mode of cell death. In addition, a combinational treatment of SFN and gemcitabine on MCF-7 cells resulted in growth inhibition in a synergistic manner with a combination index (CI) <1. Notably, SFN was found to significantly downregulate the expression of Bcl-2, an anti-apoptotic gene, and COX-2, a gene involved in inflammation, in a time-dependent manner. These results indicate that SFN induces apoptosis and anti-inflammatory effects on MCF-7 cells via downregulation of Bcl-2 and COX-2 respectively. The combination of SFN and gemcitabine may potentiate the efficacy of gemcitabine and minimize the toxicity to normal cells. Taken together, SFN may be a potent anti-cancer agent for breast cancer treatment.

Apoptosis induced by desmethyl-lasiodiplodin is associated with upregulation of apoptotic genes and downregulation of monocyte chemotactic protein-3

There is growing interest in the discovery of bioactive metabolites from endophytes as an alternative source of therapeutics. Identification of their therapeutic targets is essential in understanding the underlying mechanisms and enhancing the resultant therapeutic effects. As such, bioactive compounds produced by endophytic fungi from plants at the National Park, Pahang, Malaysia, were investigated. Five known compounds were identified using LC-UV-MS-NMR and they include trichodermol, 7-epi-brefeldin A, (3R,4S)-4-hydroxymellein, desmethyl-lasiodiplodin and cytochalasin D. The present study went on to investigate the potential anticancer effects of these compounds and the corresponding molecular mechanisms of the lead compound against human breast adenocarcinoma, MCF-7. For the preliminary screening, the cytotoxicity and apoptotic effects of these compounds against MCF-7 were examined. The compounds were also tested against noncarcinogenic hepatocytes (WRL68). The differential cytotoxicity was then determined using the MTT assay. Desmethyl-lasiodiplodin was found to suppress the growth of MCF-7, yielding an inhibitory concentration (IC50) that was seven-fold lower than that of the normal cells. The cytotoxic effect of desmethyl-lasiodiplodin was accompanied by apoptosis. Subsequent analysis demonstrated increased expression levels of caspase 3, c-myc and p53. Further, desmethyl-lasiodiplodin resulted in inhibition of monocyte chemotactic protein (MCP)-3, a cytokine involved in cell survival and metastasis. Hence, this study proposed that desmethyl-lasiodiplodin inhibited growth and survival of MCF-7 through the induction of apoptosis. This anticancer effect is mediated, in part, by upregulation of apoptotic genes and downregulation of MCP-3. As desmethyl-lasiodiplodin elicited minimal impact against normal hepatocytes, our findings also imply its potential use as a specific apoptotic agent in breast cancer treatment.