Human Breast Cell Lines Research Articles

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

Synthesis, Characterization, Antimicrobial and Antitumor Activities of Sucrose Octa(N-ethyl)carbamate.

Sucrose octa(N-ethyl)carbamate was synthesized directly from sucrose and ethyl isocyanate, and its structure was confirmed by various analytical methods, such as (1)H and (13)C NMR, FTIR, m.p., MS, and optical rotation. Its antibacterial, antifungal and cytotoxic activities were investigated. It exhibited strong inhibition against all bacteria tested, namely S. aureus (MIC 0.18±0.006), B. cereus (MIC 0.094±0.000), M. flavus (MIC 0.28±0.01), L. monocytogenes (MIC 0.18±0.006), P. aeruginosa (MIC 0.094±0.002), S. typhimurium (MIC 0.094±0.002), E. coli (MIC 0.18±0.006) and E. cloacae (MIC 0.18±0.006) and strong antifungal activity towards T. viride (MIC 0.09 ± 0.006), A. versicolor (MIC 0.18 ± 0.01), A. ochraceus (MIC 0.375 ± 0.01) and P. ochrochloron (MIC 0.375 ± 0.04). Furthermore, it showed moderate antitumor potential against human breast (GI50 357.20±14.12), colon (GI50 332.43±11.19) and cervical (GI50 282.67±3.97) cell lines and, more important, without hepatotoxicity.

Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF‐7 and MCF‐10A

Sandalwood essential oil (SEO) is extracted from Santalum trees. Although α-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2–8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p = 1.37E − 2), Ku80 (p = 5.8E − 3), EPHX1 (p = 3.3E − 3), and 14-3-3ζ (p = 4.0E − 4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells.

Phytochemicals and Cytotoxicity of Launaea procumbens on Human Cancer Cell Lines.

Background:The plant Launaea procumbens belongs to the family Asteraceae and traditionally used in the treatment rheumatism, kidney, liver dysfunctions and eye diseases. In the present study Phytochemical analysis and fractions of methanolic extract of L. procumbens leaves were tested in vitro for their cytotoxicity.Objectives:Phytochemical analysis and cytotoxic activity of methanolic extract and fractions of Launaea procumbens against four cancer cell lines K562, HeLa, MIA-Pa-Ca-2 and MCF-2 by SRB assay.Materials and Methods:Powdered leaves of Launaea procumbens were extracted sequentially with hexane, ethyl acetate, butanol and water by cold extraction. Phytochemical analysis and cytotoxicity assay were carried out for these fractions using SRB assay against four human cancer cell lines, namely leukemia (K562), cervix (HeLa), pancreatic (MIA-Pa-Ca-2) and breast (MCF-7).Results:Ethyl acetate extract exerts potent cytotoxicity against human leukemia (K562), cervix (HeLa) and breast (MCF-7) cell lines IC50 value of 25.30±0.50, 19.80±0.10 and 36.90±4.90 μg/ml respectively. Moderately cytotoxic effect found in hexane extract IC50 value of 41±8 and 48.20±0.50 μg/ml against leukemia (K562), and breast (MCF-7) cancer cell line respectively. The Chemical composition analyzed by GC-MS showed considerable differences in solvent fractions of Launaea procumbens.Conclusion:This study revealed the cytotoxic potential of ethyl acetate and hexane fractions of L. procumbens leaves on different cancer cell lines.SUMMARY Ethyl acetate and Hexane fractions of Launaea procumbens plant exhibit cytotoxicity. Among the different fractions Ethyl acetate showed relatively higher cytotoxicity.Ethyl acetate found more cytotoxic against leukemia (K 562), cervix (HeLa) and breast (MCF-7) cancer cell lines. Moderete cytotoxicity found in hexane fraction against leukemia (K 562) and breast (MCF-7) cancer cell line.GC-MS results showed L. procumbens is a rich source of 1-H- pyrazole, 1-H-imidazole, β -amyrin, α -amyrin and lupeol. These compounds may be attributed for the cytotoxic activity. Abbreviations used: SRB: Sulforhodamine B assay, MW: Molecular weight

Design and evaluation of biofunctionalized magnetic nanoparticles for biomedical applications

Event Abstract Back to Event Design and evaluation of biofunctionalized magnetic nanoparticles for biomedical applications Vera Balan1, Maria Butnaru1, Ovidiu Bredetean1 and Liliana Verestiuc1 1 Gr.T.Popa University of Medicine and Pharmacy, Faculty of Medical Bioengineering, Romania INTRODUCTION Recently, biofunctionalized magnetic nanoparticles are investigated for cancer theragnosis applications[1]. These “intelligent” particles are comprised by a magnetic core, a biocompatible surface coating, a therapeutic agent, and a recognition layer represented by a suitable receptor attached[2]. Amphiphilic derivative chitosan was used to design nanoparticles as a cancer drug delivery system with promising results regarding tumor growth inhibition[3]. Recent studies have revealed that biotin receptors are overexpressed on the surface of tumoral cells, especially breast cancer cells and imply that biotin can be used as a tumor targeting approach for various anti-cancer drugs[4]. Doxorubicin (DOX) is known as one of the most important chemotherapeutic agent against breast, ovarian or lung cancer cells. In this context, the main objective of this study was to design biofunctionalized magnetic nanostructures based on magnetite and N-palmitoyl chitosan, with dual entrapment (therapeutic agent and magnetic material) and evaluate their physico-chemical and biological characteristics. EXPERIMENTAL METHODS Biofunctionalized magnetic nanoparticles (BMNs) have been prepared in two steps: first, nanostructures based on N-palmitoyl chitosan, loaded with doxorubicin and hydrophobic magnetite have been obtained by a double emulsion method; in the next step, magnetic nanoparticles have been functionalized with biotin via carbodiimide chemistry. BMNs structure have been investigated and confirmed by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction analysis. Particles size and zeta potential were measured with a Malvern Zetasizer NanoS instrument; Magnetic properties have been evaluated using a vibrating sample magnetometer (Lakeshore VSM 7400 System) and the surface morphology was studied by transmission electron microscopy (TEM; PHILIPS CM 20). Drug loading efficiency and in vitro kinetic of drug release has been assessed by UV-VIS Spectroscopy. Internalization of biofunctionalized magnetic nanoparticles was investigated by fluorescence microscopy on MCF-7 cells (Fig 1). RESULTS AND DISCUSSION A new type of biofunctionalized magnetic nanoparticles with submicron size, positive zeta potential, dual entrapment ability, cytotoxic effects on MCF7 cell line human breast adenocarcinoma, redispersion ability and suitable magnetic properties (good magnetic saturation and superparamagnetic behaviour) have been prepared. FT-IR confirmed the structure of biofunctionalized magnetic nanocarriers: biotin, N-palmitoyl chitosan, magnetite and doxorubicin. TEM image has confirmed that hydrophobic magnetite was effectively incorporated into the magnetic nanoparticles. Fig. 1. The fluorescent microscopy image of BMNs in contact with MCF cells CONCLUSIONS These particles exhibit a multitude of good assets together with suitable biological behavior that sustain future experiments aiming to confirm the suitability of proposed magnetic nanoparticles as drug delivery system for breast cancer chemotherapy. This work was financially supported by the Romanian Ministry of Education; grant PN II- PT-PCCA-2011-3.2-0428 - INTERBIORES.

Folic acid targeted Mn:ZnS quantum dots for theranostic applications of cancer cell imaging and therapy.

In this study, we synthesized a multifunctional nanoparticulate system with specific targeting, imaging, and drug delivering functionalities by following a three-step protocol that operates at room temperature and solely in aqueous media. The synthesis involves the encapsulation of luminescent Mn:ZnS quantum dots (QDs) with chitosan not only as a stabilizer in biological environment, but also to further provide active binding sites for the conjugation of other biomolecules. Folic acid was incorporated as targeting agent for the specific targeting of the nanocarrier toward the cells overexpressing folate receptors. Thus, the formed composite emits orange–red fluorescence around 600 nm and investigated to the highest intensity at Mn2+ doping concentration of 15 at.% and relatively more stable at low acidic and low alkaline pH levels. The structural characteristics and optical properties were thoroughly analyzed by using Fourier transform infrared, X-ray diffraction, dynamic light scattering, ultraviolet-visible, and fluorescence spectroscopy. Further characterization was conducted using thermogravimetric analysis, high-resolution transmission electron microscopy, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray fluorescence, and X-ray photoelectron spectroscopy. The cell viability and proliferation studies by means of MTT assay have demonstrated that the as-synthesized composites do not exhibit any toxicity toward the human breast cell line MCF-10 (noncancer) and the breast cancer cell lines (MCF-7 and MDA-MB-231) up to a 500 µg/mL concentration. The cellular uptake of the nanocomposites was assayed by confocal laser scanning microscope by taking advantage of the conjugated Mn:ZnS QDs as fluorescence makers. The result showed that the functionalization of the chitosan-encapsulated QDs with folic acid enhanced the internalization and binding affinity of the nanocarrier toward folate receptor-overexpressed cells. Therefore, we hypothesized that due to the nontoxic nature of the composite, the as-synthesized nanoparticulate system can be used as a promising candidate for theranostic applications, especially for a simultaneous targeted drug delivery and cellular imaging.

MicroRNA-153 Regulates NRF2 Expression and is Associated with Breast Carcinogenesis.

Abnormal expression of NRF2 levels in some tumor tissues may enhance drug resistance through various mechanisms. Recent studies have demonstrated a link between dysregulated expression of miRNAs and breast carcinogenesis. Quantitative RT-PCR analysis was used to examine the expression levels of miR-153 in breast cancer cell lines. The biological effects of miR-153 were assessed in CRC cell lines using clonogenic cell survival assay, mammosphere formation assay, cell migration assay, 8-OHdG estimation assay, and flow cytometry analysis. Quantitative RT-PCR and western blot analyses were employed to evaluate the expression of miR-153 targets. In this study, we investigated the role of vitamin C in the regulation of miR-153 (miR-153) and its target gene(s) in cell models of mammary carcinogenesis. In human breast cell lines treated with E2, the level of miR-153 was found to be increased. In contrast, vitamin C treatment was able to decrease the expression of miR-153. Bioinformatic prediction indicates nuclear factor erythroid 2-related factor 2 (NRF2) may be a target for miR-153. In E2-treated breast cancer cell lines, NRF2 protein level was found to be decreased. Overexpression of miR-153 significantly reduced NRF2 and the downstream genes. Furthermore, miR-153 was found to decrease apoptosis and increase colony formation in breast epithelial cells. These data indicate that miR-153 acts as an oncogene in breast carcinogenesis by targeting NRF2.

Design, Synthesis and Biological Evaluation of Novel Class Diindolyl Methanes (DIMs) Derived from Naturally Occurring Phenolic Monoterpenoids

Several Diindolyl alkanes and their derivatives have been isolated from plant and marine sources. Among the various derivatives of indoles, Diindolyl methanes have wide medicinal applications such as to induce apoptosis in human cancer cells, antibacterial, Anti-inflammatory, antiviral and hormonal control activities. Therefore, they play essential role in marine as well as terrestrial living systems. In present studies we report novel class of Diindolyl methanes prepared from natural phenolic monoterpenoids, via ortho formylation of phenolic monoterpenoids (Carvacrol, Thymol and Eugenol), followed by synthesis, characterization, anticancer, antioxidant and α-amylase inhibitory activities. All the synthesized derivatives show moderate anticancer activities against human breast cell line MCF7, good antioxidant and α-amylase inhibitory activities using DPPH and α-amylase assay respectively.

Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation

Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16β-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone.The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65μM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4μM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Abstract A140: Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras

Abstract Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Through iterative chemical synthesis and screening using a phenotypic assay designed to select for Ras inhibitors, we identified a small-molecule lead compound that was then optimized for potency and selectivity to inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras. Materials and methods: Viable cell number was measured using the CellTiter-Glo® ATP assay (Promega) following 72 hr of treatment. Ras activation status was measured by precipitating GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose followed by western blotting using anti-Ras antibody. Ras binding was determined by pre-incubating cell lysates with GST-Raf1-RBD/GSH sepharose, treating with test compounds for 30 min, followed by western blotting using anti-Ras antibody. Cell cycle distribution was measured by DNA content following DyeCycle Green labeling. Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines harboring activated K-Ras, N-Ras or H-Ras with selectivity indices greater than 100-fold over cells lacking activated Ras. By surveying a large panel of human colon, breast, and lung tumor cell lines, a strong correlation was measured between potency to inhibit tumor cell growth and Ras activation status. Ras selectivity was confirmed by transfecting human H322 lung tumor cells that lack activated Ras with mutant H-Ras and inducing sensitivity to DC070-547. The compound inhibited Ras-Raf binding as shown by Ras-pull down in cell lysates following treatment with DC070-547 at concentrations that inhibit tumor cell growth. DC070-547 also caused cell cycle arrest in the G2 phase selectively in tumor cells containing activated Ras. Interestingly, cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment. Conclusion: While Ras is widely considered to be non-druggable, we have identified a series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras. With promising drug-like properties, DC070-547 has been selected from this series as a prospective drug development candidate that is being evaluated for anti-tumor efficacy and toxicity in preclinical models. Citation Format: Gary A. Piazza, Bing Zhu, Kevin Lee, Joshua Canzoneri, Sara Sigler, Ashley Lindsey, Veronica Ramirez-Alcantara, Luciana Madeira da Silva, Haddon Mullins, Alisa Trinh, Kristy Berry, Jacob Valiyaveettil, Adam B. Keeton, Xi Chen, Michael R. Boyd. Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A140.

Corrigendum: In-vitro effects of protease inhibitors on BAX, BCL-2 and apoptosis in two human breast cell lines

Corrigendum: In-vitro effects of protease inhibitors on BAX, BCL-2 and apoptosis in two human breast cell lines

In-vitro effects of protease inhibitors on BAX, BCL-2 and apoptosis in two human breast cell lines (with corrigendum)

Currently, the treatment of choice of HIV/AIDS in South Africa is the multidrug combination regimen known as HAART (highly active antiretroviral treatment). HAART, which commonly consists of nucleoside or non-nucleoside reverse transcriptase inhibitors and protease inhibitors, has radically decreased mortality and morbidity rates among people living with HIV/AIDS. The emphasis of the original development of the antiretroviral drugs was on clinical effectiveness (reducing mortality). Presently, emphasis has shifted from the initial short- term considerations to the long-term undesirable or harmful effects induced by this treatment regimen. Whether antiretroviral compounds are oncogenic is widely speculated, which led to this investigation into the effects of protease inhibitors on the expression of key apoptotic regulatory genes, BAX and BCL-2, in two human breast cell lines, MCF-7 and MCF-10A by real-time qPCR gene expression and immunofluorescence. The anti-apoptotic effects of the protease inhibitors – LPV/r were also investigated by cell death detection ELISA and acridine orange staining. This study also evaluated the cytotoxicity of the antiretroviral drugs in normal and cancer cell lines of the breast (at clinically relevant concentrations of the drugs and at different time points, 24–96 h), employing the neutral red uptake assay. The drugs and combinations tested did not alter BAX and BCL-2 gene expression and protein expression and localisation in both cell lines. In addition, the protease inhibitors–LPV/r did not inhibit camptothecin-induced apoptosis in both cell lines. We have shown that the protease inhibitors demonstrated varying degrees of cytotoxicity in the breast cells. The resulting DNA damage associated with cytotoxicity is strongly implicated in the processes of tumour initiation.

Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

Effect of processing on the content and biological activity of polysaccharides from Pleurotus ostreatus mushroom

Water soluble polysaccharides (WSP) were isolated from processed and non-processed fruiting bodies of oyster mushroom (Pleurotus ostreatus). The processing methods involved: blanching, boiling and blanching followed by fermenting with a strain of lactic acid bacteria (Lactobacillus plantarum). The yields of WSP ranged from 78.7 ± 1.5 mg/g to 120.1 ± 4.9 mg/g dry weight of sample. Blanching did not affect the content of WSP. Boiling for 15 min, led to the substantial decrease in the amount of WSP (34.7% decline). The isolated samples differed in carbohydrate, protein and phenolics content. FTIR spectroscopy of the WSP samples confirmed the presence of both α- and β-glycosidic linkages. Gel permeation chromatography showed the presence of compounds having the molecular weight of 198.3, 11.9, 3.1 kDa. The samples possessed antioxidant capacity measured by ABTS method (14.14 ± 0.63 to 29.48 ± 1.12 μmoles of Trolox per 1 g dw) and FRAP assay (2.49 ± 0.54 to 16.52 ± 0.55 μmoles of Trolox equivalents per 1 g dw). The antioxidant potential was decreased by the processing. Similarly, antiproliferative activity of WSP towards human breast cell lines (MCF-7 and T-47D) was lower due to the processing.

RECK is not an independent prognostic marker for breast cancer.

BackgroundThe REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear.MethodsThe RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated.ResultsHigher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables.ConclusionsAlthough it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients.

The synergistic effect between celecoxib and luteolin and dependence on estrogen receptor in human breast cancer cells.

135 Background: The anti-cancer effects of celecoxib and luteolin are well known. Although our previous study demonstrated that the combination of celecoxib and luteolin synergistically inhibits breast tumor growth compared with each of the treatments alone, we did not uncover the molecular mechanisms of these effects. The aims of our present study were to compare the effects of a celecoxib and luteolin combination treatment in four different human breast cell lines and to determine the mechanisms of action in vitro and in vivo. Methods: Using MCF-7, MCF7/HER18, MDA-MB-231 and SkBr3 human breast cancer cells, proliferation assay, apoptosis assay, inhibition assay with MEK and PI3K inhibitor in addition to western blotting and xenograft study after treatment with celecoxib and luteolin. Results: The synergistic effects of a celecoxib and luteolin combination treatment yielded significantly greater cell growth inhibition in all four breast cancer cell lines compared with the single agents alone. In particular, combined celecoxib and luteolin treatment significantly decreased the growth of MDA-MB-231 cancer cells in vivo compared with either agent alone. The celecoxib and luteolin combination treatment induced synergistic effects via Akt inactivation and extracellular signal-regulated kinase (ERK) signaling inhibition in MCF-7 and MCF7/HER18 cells and via Akt inactivation and ERK signaling activation in MDA-MB-231 and SkBr3 cells. Conclusions: These results demonstrate the synergistic anti-tumor effect of the celecoxib and luteolin combination treatment in different four breast cancer cell lines, thus introducing the possibility of this combination as a new treatment modality.

Abstract PR01: Long noncoding RNA PVT1 potentiates MYC in human cancers with 8q24 gain

Abstract Copy number gain of the 8q24.21 chromosomal region have been a prominent mutation in many human cancers and are associated with poor prognosis. The well-characterized MYC oncogene, which resides in the 8q24.21 region, has been, conventionally, the attractive candidate driving these cancers. However, MYC is often co-gained with an adjacent ”gene desert” region. This “gene desert” often encompasses the long non-coding RNA gene PVT1, the CCDC26 gene candidate, and the GSDMC gene. Whether low copy number gain of one or more of these genes drives neoplasia is not known. We used chromosome engineering in mice to develop strains containing an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced into these three strains to test for changes in latency and penetrance in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc, (but not those with an extra copy of Myc or Pvt1,Ccdc26,Gsdmc), developed adenocarcinomas with reduced latency, suggesting that while an additional copy of the Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the mouse mammary tumors, as well as in two human breast cell lines with 8q24 amplification (SK-BR-3 and MDA-MB-231). Silencing of PVT1 markedly decreased MYC protein levels, while no effect was seen in MYC transcript levels, suggesting a PVT1-dependence of MYC protein even in high MYC- amplification cancer cells. Analysis of the TCGA and Progenetix databases suggested that 18% of 45,922 human tumors queried harbor MYC copy number increases, and in >99% of MYC-copy-increase cancers, copy number of PVT1 was co-increased .Tissue microarray analysis using in situ hybridization for PVT1 and immunohistochemistry for MYC revealed that PVT1 RNA and MYC protein expression correlated in primary human tumors. These data together suggested that PVT1 can potentiate MYC in human cancer. CRISPR mediated deletion of PVT1 in colorectal cancer cell line HCT116 either partially or fully ablated tumor formation in xenografts and significantly reduced MYC protein levels by ~50%. We propose that the dependence of high MYC protein levels on PVT1 lncRNA provides a much-needed therapeutic target against MYC protein, which has been refractory to small molecule inhibition. Citation Format: Yuen-Yi Tseng, Anindya Bagchi. Long noncoding RNA PVT1 potentiates MYC in human cancers with 8q24 gain. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR01.

Combined effect of silver nanoparticles and other cosmetic additives on viability of human breast cell lines

Combined effect of silver nanoparticles and other cosmetic additives on viability of human breast cell lines

Health-benefits of date fruits produced in Saudi Arabia based on in vitro antioxidant, anti-inflammatory and human tumor cell proliferation inhibitory assays

Date fruits are reported to exhibit health-beneficial effects in addition to its nutritional value. Fruits collected from commercial date palm trees were sequentially extracted with water and methanol. All varieties of date fruits contained sugars, phenolics, triterpenoids, triglycerides, fatty acids and steroids, where sugars were the predominant components. Water and methanolic extracts of date fruits were assayed for antioxidant, antiinflammatory and human tumor cell proliferation inhibitory activities. In MTT antioxidant assay, methanolic extracts at 250μg/mL exhibited moderate activity with absorbance values between 0.14 and 0.41. Water and methanolic extracts at 100μg/mL inhibited lipid peroxidation (LPO) by 50–67% and 58–82%, respectively. In anti-inflammatory assay using cyclooxygenase enzymes (COX-1 and -2), water and methanolic extracts at 100μg/mL showed COX-1 enzyme inhibition by 26–36% and 33–41%, and COX-2 by 45–48% and 48–52%, respectively. At 100μg/mL concentrations, methanolic extracts of all date fruits showed marginal cell proliferation inhibitory activity against human gastric, prostate, colon, breast and lung tumor cell lines. The bioassay results suggest that varietal difference is not a significant factor among the 29 date fruits studied when compared for health-beneficial effects resulting from non-nutritional components present in it.