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Fibrous sheath interacting protein 1 (FSIP1), a spermatogenesis-related testicular antigen, is expressed in abundance in breast cancers, particularly in those overexpressing human epidermal growth factor receptor 2 (HER2); however, little is known about its role in regulating the growth and metastasis of breast cancer cells. We and others have shown previously that FSIP1 expression in breast cancer correlates positively with HER2-positivity, recurrence, and metastases and negatively with survival. Here, using coimmunoprecipitation and microscale thermophoresis, we find that FSIP1 binds to the intracellular domain of HER2 directly. We further show that shRNA-induced FSIP1 knockdown in SKBR3 and MCF-7 breast cancer cells inhibits proliferation, stimulates apoptosis, attenuates epithelial–mesenchymal transition, and impairs migration and invasiveness. Consistent with reduced proliferation and enhanced apoptosis, xenotransplantation of SKBR3 cells stably transfected with sh-FSIP1 into nu/nu mice results in reduced tumor volumes compared with sh-NC transplants. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping using sh-FSIP1 gene signature yielded associations with extracellular matrix protein pathways, and a reduction in SNAI2 protein expression was confirmed on Western blot analysis. Complementarily, interrogation of the Connectivity Map using the same gene signature yielded, as top hits, chemicals known to inhibit epithelial–mesenchymal transition, including rapamycin, 17-N-allylamino-17-demethoxygeldanamycin, and LY294002. These compounds phenocopy the effects of sh-FSIP1 on SKBR3 cell viability. Thus, FSIP1 suppression limits oncogenesis and invasiveness in breast cancer cells and, considering its absence in most other tissues, including normal breast, may become a potential target for breast cancer therapy.

Cancer treatment has been very challenging in recent decades. One of the most promising cancer treatment methods is hyperthermia, which increases the tumor temperature (41–45 °C). Magnetic nanoparticles have been widely used for selective targeting of cancer cells. In the present study, magnetic dextran-spermine nanoparticles, conjugated with Anti-HER2 antibody to target breast cancer cells were developed. The magnetic dextran-spermine nanoparticles (DMNPs) were prepared by ionic gelation, followed by conjugation of antibody to them using EDC-NHS method. Then the Prussian blue method was used to estimate the targeting ability and cellular uptake. Cytotoxicity assay by MTT showed that antibody-conjugated MNPs (ADMNPs) have no toxic effect on SKBR3 and human fibroblast cells. Finally, the hyperthermia was applied to show that synthesized ADMNPs, could increase the cancer cells temperature up to 45 °C and kill most of them without affecting normal cells. These observations proved that Anti-HER2 conjugated magnetic dextran-spermine nanoparticles can target and destroy cancer cells and are potentially suitable for cancer treatment.

Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer using three different imaging modalities. These findings warrant further development of this activated platelet specific scFvGPIIb/IIIa, potentially as a universal marker for cancer diagnosis and ultimately for drug delivery in an innovative theranostic approach.

In this article, we demonstrate the use of bio-conjugated 2D graphene oxide (bio-GO) nanostructure to probe breast cancer cell (SKBR3) with excellent discrimination over other types of circulating tumor cells. We distinctly observed that bio-GO nanostructure targets and bind SKBR3 cell selectively in the cell mixture. Longer incubation of SKBR3 cell with bio-GO causes Raman signal "turn off" when excited with 532 nm laser. This is attributed to penetration of the bio-GO through the plasma membrane of the cell by generating transient hole. Extraction of GO after cell digestion also support the internalization rubric of 2D graphene through cell membrane. Our experimental data with the HaCaT healthy cell line, as well as with LNCaP prostate cancer cell line clearly demonstrated that this Raman scattering assay is highly selective to SKBR3. The mechanism of selectivity and the assay's response change have been verified and discussed utilizing fluorescence properties of GO and various other techniques. The experimental results open up a possibility of new label free Raman scattering assay, for reliable diagnosis of cancer cell lines by monitoring "turn-off" of the Raman signal from Bio-GO nanostructure.

BACKGROUND Toll-like receptor 9 (TLR9) is a DNA receptor of innate immune system which plays a pivotal role in inflammatory response. Recent evidence reveals over-expression and functionality of TLR9 in a wide variety of cancer cells and its contribution to tumor cell proliferation and survival. OBJECTIVE In this study, we assessed the aberrant cell surface expression of TLR9 in cancer using cell-lines model. METHODS Three breast cancer cell-lines (MDA-MB-231, MCF7 and SKBR3) and five colorectal adenocarcinoma cell-lines (HT29, HT29/219, SW480, SW48 and SW1116) in addition to one primary foreskin isolated fibroblast cell were analyzed for cell surface and intracellular expression of TLR9 by flow cytometry method. RESULTS Maximum surface expression of TLR9 was observed in colorectal cell-line HT29/219 (38.35%), as compared with the bottom line fibroblast normal cells (0.12%). The most intracellular expression was observed in MCF-7 cells (35.63%), whereas MDA-MB-231 expressed the maximum surface/intra cellular expression (277 times). CONCLUSIONS Based on the results, we hypothesize that aberrant surface expression of TLR9 on tumor cells may promote tumor growth and invasion. It might also highlight a dual contradictory role for CpG-ODNs, as adjutant in cancer therapy.

Two-deoxy-D-glucose (2DG) causes cytotoxicity in the cancer cells by disrupting the thiol metabolism, and MLN4924 inactivates the SCF E3 ligase and so causes the accumulation of its substrates which trigger apoptosis and hence might enhance the efficiency of radiotherapy and overcame on the radioresistance of the cancer cells.SKBR3 and MCF-7 breast cancer cells were treated with 500 μM 2DG and/or MLN4924 (30, 100, 200 and 300 nM), and in combination in the presence and absence of 1, 1.5 and 2 Gy gamma irradiation. The effects of the treatments - 2DG, MLN4924, irradiation alone and combined - on MCF-7 and SKBR3 cell lines were evaluated by MTT assay, TUNEL assay, cell death detection, Q-PCR for caspase-3 and Bcl-2 expression analysis, and finally clonogenic survival assay.The treatments enhanced the further radio cytotoxicity via inducing the apoptosis cell signaling gene, caspase-3. The 2DG and MLN4924 treatments could act as a radiosensitizer, especially on the SKBR3 cells, and further sensitized the cells with a sensitivity enhancement ratio (SER) of 1.41 and 1.27 in SKBR3 and MCF-7 cells, respectively.The combined chemo-radiotherapy might improve the breast cancer treatment outcome.

Hyaluronan (HA), a polymer with various molecular weights (MW) found in tumor microenvironments, is associated with malignant progression of breast cancer. Reducing the amount of high-MW HA in the microenvironment by hyaluronidase is a promising approach for breast cancer treatment. However, whether the generation of HA fragments negatively affects breast cancer cells remains to be determined. Furthermore, HA forms three-dimensional (3D) networks by cross-linking with other extracellular molecules to function. Therefore, a model mimicking the cross-linked HA network is required to determine the effect of HA fragments on breast cancer cells. To clarify the differential roles of low (HA35) versus high (HA117) MW HA on cancer cell phenotype, a 3D culture system was set up by covalently cross-linking HA with alginate and investigating the behavior of 4T-1 and SKBR3 breast cancer cells alongside a two-dimensional (2D) control. The results show the invasion and migration abilities of 4T-1 and SKBR3 cells are significantly enhanced by the presence of HA35 but inhibited by HA117 in both 2D monolayers and 3D spheroids. The differential effects of HA35 and HA117 on cancer cell epithelial-mesenchymal transition (EMT) phenotype were further confirmed in terms of differential regulation of E-cadherin and vimentin as important EMT markers at both the cellular and mRNA levels. Additional experiments show the CD44-Twist signaling pathway might be involved in the differential effects of HA35 and HA117. These results have important implications with respect to understanding the role of HA in breast cancer development and for the design of therapeutic approaches based on the eradication of HA with hyaluronidase.

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2′-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell–based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

A phenaxazone compound [5H-Benzo[a]phenoxazin-5-one (BP)] along with an aminoquinone[2-[(o-hydroxyphenyl)amino]-1,4-naphthaquinone (HAN)] derivatives were synthesized from lawsone using ultrasound irradiation technique. The structure of the compounds were characterized by elemental analysis and various spectral studies. Optoelectronic properties were studied using Schrodinger material science suit (2015). The compounds exhibit fluorescence emission in longer wave length it may find applications in photodynamic therapy. Single crystal X-ray diffraction studies reveals that the compound BP crystallizes in monoclinic space group. The antioxidant activity of HAN and BP were determined using DPPH radical scavenging assay and the results indicate that both the compounds have good antioxidant capacity, HAN having more scavenging activity than BP. Lead molecules were identified using in silico molecular docking studies as a green chemistry approach. iGEMDOCK, GOLD and Schrodinger softwares were used for these studies. The docking studies reveal that the structural modification of the parent compound gave more active compounds making them promising lead molecules. The lead molecules were subjected to in vitro studies. The cytotoxicity of BP and HAN was studied using human breast cancer (SKBR3) cell lines. The IC50 value of HAN was found to be 19.8μM while BP was found to have cell viability, less than 10% even at 25μM concentration. The chemotherapeutic agents kill the cancer cells mainly through apoptosis. HAN and BP were subjected to apoptosis studies. BP was found to more active than HAN. Thus it can be suggested that the mechanism of cell death may be through apoptosis.

Summary B55α is a regulatory subunit of the PP2A phosphatase. We have recently found that B55α-associated PP2A promotes partial deactivation of the HIF-prolyl-hydroxylase enzyme PHD2. Here, we show that, in turn, PHD2 triggers degradation of B55α by hydroxylating it at proline 319. In the context of glucose starvation, PHD2 reduces B55α protein levels, which correlates with MDA-MB231 and MCF7 breast cancer cell death. Under these conditions, PHD2 silencing rescues B55α degradation, overcoming apoptosis, whereas in SKBR3 breast cancer cells showing resistance to glucose starvation, B55α knockdown restores cell death and prevents neoplastic growth in vitro. Treatment of MDA-MB231-derived xenografts with the glucose competitor 2-deoxy-glucose leads to tumor regression in the presence of PHD2. Knockdown of PHD2 induces B55α accumulation and treatment resistance by preventing cell apoptosis. Overall, our data unravel B55α as a PHD2 substrate and highlight a role for PHD2-B55α in the response to nutrient deprivation., Graphical Abstract, Highlights • PHD2 hydroxylates and degrades B55α, a regulatory subunit of PP2A phosphatase • In the absence of glucose, a high αKG-to-fumarate ratio favors PHD2 activation • Active PHD2 degrades B55α and promotes cell death under glucose starvation • PHD2-silenced xenografts showed accumulation of B55α and resistance to 2DG treatment, Di Conza et al. show that, following glucose starvation, a high αKG-to-fumarate ratio favors PHD2 activation that promotes apoptosis by degrading B55α. In breast cancer cells, PHD2 knockdown prevents B55α degradation and overcomes apoptosis in response to glucose starvation. PHD2-silenced MDA-MB231 xenografts show accumulation of B55α and resistance to 2DG treatment.

Cancer has proceeded to surpass one of the most chronic illnesses to be the major cause of mortality in both the developing and developed world. Garcinia mangostana L. (mangosteen, family Guttiferae) known as the queen of fruits, is one of the most popular tropical fruits. It is cultivated in Southeast Asian countries: Malaysia, Indonesia, Sri Lanka, Burma, Thailand, and Philippines. Traditionally, numerous parts of G. mangostana have been utilized to treat various ailments such as abdominal pain, haemorrhoids, food allergies, arthritis, leucorrhoea, gonorrhea, diarrhea, dysentery, wound infection, suppuration, and chronic ulcer.Although anticancer activity has been reported for the plant, the goal of the study was designed to isolate and characterize the active metabolites from G. mangostana and measure their cytotoxic properties. In this research, the mechanism of antiproliferative/cytotoxic effects of the tested compounds was investigated.The CHCl3 fraction of the air-dried fruit hulls was repeatedly chromatographed on SiO2, RP18, Diaion HP-20, and polyamide columns to furnish fourteen compounds. The structures of these metabolites were proven by UV, IR, 1D, and 2D NMR measurements and HRESIMS. Additionally, the cytotoxic potential of all compounds was assessed against MCF-7, HCT-116, and HepG2 cell lines using SRB-U assay. Antiproliferative and cell cycle interference effects of potentially potent compounds were tested using DNA content flow cytometry. The mechanism of cell death induction was also studied using annexin-V/PI differential staining coupled with flow cytometry.The CHCl3 soluble fraction afforded two new xanthones: mangostanaxanthones V (1) and VI (2), along with twelve known compounds: mangostanaxanthone IV (3), β-mangostin (4), garcinone E (5), α-mangostin (6), nor-mangostin (7), garcimangosone D (8), aromadendrin-8-C-β-D-glucopyranoside (9), 1,2,4,5-tetrahydroxybenzene (10), 2,4,3`-trihydroxybenzophenone-6-O-β-glucopyranoside (11), maclurin-6-O-β-D-glucopyranoside (rhodanthenone) (12), epicatechin (13), and 2,4,6,3`,5`-pentahydroxybenzophenone (14). Only compound 5 showed considerable antiproliferative/cytotoxic effects with IC50's ranging from 15.8 to 16.7µM. Compounds 3, 4, and 6 showed moderate to weak cytotoxic effects (IC50's ranged from 45.7 to 116.4µM). Using DNA content flow cytometry, it was found that only 5 induced significant cell cycle arrest at G0/G1-phase which is indicative of its antiproliferative properties. Additionally, by using annexin V-FITC/PI differential staining, 5 induced cells killing effect via the induction of apoptosis and necrosis in both HepG2 and HCT116 cells. Compound 3 produce necrosis and apoptosis only in HCT116 cells. On contrary, 6 induced apoptosis and necrosis in HepG2 cells and moderate necrosis in HCT116 cells.Fourteen compounds were isolated from chloroform fraction of G. mangostana fruit hulls. Cytotoxic properties exhibited by the isolated xanthones from G. mangostana reinforce the avail of it as a natural cytotoxic agent against various cancers. These evidences could provide relevant bases for the scientific rationale of using G. mangostana in anti-cancer treatment.

miRNAs have been shown to be involved in breast cancer growth and progression. miR-4262 is a potential tumor promoter in human cancers. In this study, we first investigated the role of miR-4262 in the proliferation and invasion of human breast cancer cells. Our results showed that, compared with the adjacent tissues and MCF-10A normal breast epithelial cells, miR-4262 was markedly increased in the breast cancer tissues and five cell lines, including MDA-MB-231, MDA-MB-468, MDA-MB-435, SKBR3, and MCF-7. Then the miR-4262 mimic or oligo anta-miR-4262 was transfected into MDA-MB-231 and MCF-7 breast cancer cell lines. The results showed that the miR-4262 mimic greatly increased the miR-4262 level and the proliferation and invasion of MDA-MB-231 and MCF-7 cells. In contrast, the anta-miR-4262 had a completely opposite effect on miR-4262 expression, cell proliferation, and cell invasion in MDA-MB-231 and MCF-7 cells. Moreover, bioinformatics and luciferase reporter gene assays confirmed that miR-4262 targeted the mRNA 3′-UTR region of KLF6 and KLF15, two characterized tumor suppressor genes. miR-4262 suppressed protein levels of KLF6 and KLF15 in MDA-MB-231 cells, and the suppression could be rescued by the transfection of pcDNA-KLF6 and -KLF15. In conclusion, miR-4262 positively regulates proliferation and invasion of human breast cancer cells via suppression of KLF6 and KLF15.

Alternative cleavage and polyadenylation (APA) is involved in several important biological processes in animals, e.g. cell growth and development, and cancer progression. The increasing data show that cancer cells are inclined to produce mRNA isoforms with a shortened 3'UTR undergoing APA. For example, the Dicer1 isoform with a shorter 3'untranslated region (3'UTR) was found to be overexpressed in some cancer cells, which may be used as a potential novel prognostic biomarker for cancer. In the present work, a novel electrochemical biosensor for ultrasensitive determination of Dicer1 was designed by using gold nanoparticles and p-sulfonated calix[6]arene functionalized reduced graphene oxide (Au@SCX6-rGO) as nanocarriers. The results showed that the expressions of the shorter 3'UTR (Dicer1-S) both in BT474 and SKBR3 were obviously higher than those of the longer Dicer1 (Dicer1-L) by the constructed biosensor, which agreed well with the result analyzed by the RT-qPCR method. The detection ranges of Dicer1-S and Dicer1-L were 10-14-10-9 M and 10-15-10-10 M. The LODs were 3.5 and 0.53 fM. The specificity of the proposed biosensor was also very high. For the first time, the expressional analysis of different 3'UTRs caused by APA was studied by an electrochemical method. Moreover, the use of a macrocyclic host for constructing an electrochemical/biosensing platform has rarely been reported. The proposed electrochemical sensing strategy is thus expected to provide a new method for determination of novel biomarkers and a novel method for fast and cheap analysis of APA.

Cell cycle control is a promising target in cancer treatments, and some small-molecule cyclin-dependent kinase (CDK) inhibitors have exhibited clinical effectiveness. However, no biomarkers predictive of efficacy have been developed. Recent studies have revealed that CDK inhibitor (CKI) proteins, such as p27 and p16, also induced cytoprotective autophagy in cancer cells. However, it is unclear whether small-molecule CKIs also induce autophagy in solid tumors, as induced autophagy promotes cancer cell survival. In this study, we revealed that a CDK4 inhibitor and a CKI with a broad range of targets (flavopiridol) induced autophagy in some, but not all, solid cancer cell lines. Autophagy induction by CDK4 inhibitor was observed in BT474, MDA-MB435S, SKBr3 (derived from breast cancer), A431 (derived from epidermoid cancer), and SW480 (derived from colorectal cancer) cell lines. No such autophagy was observed in MCF7, MDA-MB231 (derived from breast cancer), NCI-N87 (derived from gastric cancer), and KMST-6 (derived from a fibroblast). In the cell lines showing autophagy, which was induced by CDK4 inhibitor, the combination of CDK4 inhibitor and autophagy inhibition by either chloroquine (CQ) or knockdown of ATG5 or BECN1 induced apoptosis. However, it did not induce apoptosis in the cell lines in which autophagy was not induced by CDK4 inhibitor. These findings indicate that the autophagy induced by CDK4 inhibitor mimics stress-induced autophagy in some solid cancer cell lines. The combination of a small-molecule CKI involved in G1/S arrest and an autophagy inhibitor leads to a synthetic lethal interaction and could become a new antitumor strategy for solid tumors showing cytoprotective autophagy induced by small-molecule CKIs.

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

The synthesis of two series of novel substituted pyrimidine derivatives bearing a sulfamide group have been described and their in vitro cancer growth inhibition activities have been evaluated against three human tumour cell lines (HT-29, M21, and MCF7). In general, growth inhibition activity has been enhanced by the introduction of a bulky substituent on the aromatic ring with the best compound having GI50

A novel peptide (P75) targeting EGFR and HER2 is successfully screened from a one-bead-one-compound (OBOC) library containing approximately 2 × 105 peptides built with the aid of computational simulation. In vitro and in vivo analyses show that P75 binds to human epithelial growth factor receptor (EGFR) with nanomolar affinity and to epithelial growth factor receptor-2 (HER2) with a lower affinity but comparable to other reported peptides. The peptide is used to modify the surface of magnetosome nanoparticles (NPs) for targeted magnetic resonance imaging (MRI). In vitro and in vivo fluorescence imaging results suggest peptide P75 modified magnetosomes (Mag-P75) specifically bind to MDA-MB-468 and SKBR3 cells as well as xenograft tumors with surprisingly low accumulation in other organs including liver and kidney. In vivo T2-weighted MR imaging studies of the xenograft tumors from SKBR3 and MDA-MB-468 cells show obviously negative contrast enhancement. The high affinity and specificity of P75 to EGFR and HER2 positive tumors, together with the success of peptide functionalized magnetosome NPs for targeted MRI demonstrate the potential of this peptide being used in the EGFR and HER2 positive tumors diagnosis and therapy.

Neo-tanshinlactone (NTL) a natural product is known for its specificity and selectivity towards the breast cancer cells. By NTL D-ring modification approach, 13 new analogues were synthesized (1A-1M). Among them 1J showed the best anticancer activity in MCF-7 (ER+, PR+/-, HER2-), SKBR3 (ER-, PR-, HER2+) and MDA-MB-231 (ER-, PR-, HER2-) cells lines with IC50 value 11.98nM, 23.71nM, and 62.91nM respectively. 1J showed minor grove binding interaction with DNA at AT-rich region and induced DNA double strand breaks (DDSBs). This had triggered several key molecular events involving, activation of ATM, Chk2 and p53, reduction in mitochondrial potential (Δψm) leading to caspase-3 and PARP cleavage mediated apoptosis. These results along with other biochemical studies strongly suggest that novel NTL analogue 1J caused DNA cleavage mediated apoptosis in the breast cancer cells and this may serve as potential lead for future breast cancer treatment.

Background: Although advances in neoadjuvant and adjuvant treatments have led to enhanced survival in patients with HER2 positive breast cancer, intraoperative detection of residual disease and tumor margins (including HER2 positive DCIS) remain challenging. Real-time fluorescence imaging is being used during surgery to improve tumor visualization and identify margins. However, the lack of an approved intraoperative imaging agent that is specific for HER2 expressing cancers limits the utility of fluorescence-guided surgery (FGS) in this patient population. We examined the potential of using clinically approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, for the development of fluorescent immunoconjugates dual-labeled with radioactivity to enable quantitative assessment. Methods: Trastuzumab, and pertuzumab were conjugated to the near-infrared (NIR) fluorophore, IR800CW, and radiometal chelator, deferoxamine (DFO), to permit dual labeling with 89Zr (positron emitting radionuclide). To confirm retention of HER2 specificity and functionality, flow cytometry analysis of the dual-labeled immunoconjugates was performed in HER2+ (BT474, SKBR3), HER2 moderate (MCF7), and HER2− (MDA231) cell lines. Using the same cell lines, radioactive uptake studies were employed to quantitatively measure cellular uptake study using 89Zr with and without blocking doses of unlabeled mAb. In vivo optical imaging of each immunoconjugate and IgG controls was performed in BT474 and MCF7 xenografts in BALB/c (nu/nu) mice 48 hours post injection using the BrukerXtreme system. Results: In vitro results confirmed retention of receptor mediated binding on both flow cytometry and radioactive analyses. As shown in Table 1, uptake of each immunoconjugate correlated with the degree of HER2 expression. Importantly, the addition of unlabeled mAb led to a 79-93% reduction in binding to cells with the highest HER2 expression (BT474 and SKBR3). In MDA231 cells binding of the immunoconjugates was similar to the IgG control and indicates low non-specific binding. In vivo and ex vivo analysis of HER2+ and HER2 moderate xenografts demonstrated prominent tumor uptake with minimal signal in non-clearance organs. As a result, tumor-to-muscle, tumor-to-fat pad, and tumor-to-lung ratios were >5, indicating excellent contrast in key regions where surgical excisions are performed clinically. Table 1: Uptake of 89Zr-mAb-IR800 in breast cancer cell linesCell Lines% Uptake of 89Zr-PertuzumabPertuzumab Block (Unlabeled)% Uptake of 89Zr-TrastuzumabTrastuzumab Block (Unlabeled)% Uptake of 89Zr-IgGBT47449.2±14.83.7±0.036.9±5.84.6±2.61.3±0SKBR337.9±1.77.9±0.942.8±3.52.9±1.73.5±0.4MCF721.7±2.63.2±0.44.0±0.12.5±0.11.3±0.7MDA2311.9±0.40.8±0.31.0±0.31.9±0.51.3±0.7 Conclusion: This study demonstrates the potential utility of HER2-targeted FGS to improve tumor visualization, increase the likelihood of obtaining clean surgical margins, and ultimately reduce morbidity in patients with HER2 breast cancer. Citation Format: Jo Simien, Julie Voss, A.M. Thompson, S.C. Ghosh, S. AghaAmiri, A. Azhdarinia, H.S. Tran-Cao. HER2/neu antibody-fluorophore conjugate for intraoperative detection of HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-01.

CpG-oligodeoxynucleotide (CpG-ODN) can function as an immune adjuvant. Previously, we showed that stimulation of breast cancer cells with CpG-ODN conjugated with PAMAM dendrimer-coated magnetic nanoparticles (DcMNPs) has induced apoptosis. The aim of the current study was to evaluate the expression levels of some apoptosis-regulating genes in several human breast cancer cells treated with CpG/DcMNPs. Treated MDA-MB231 cells showed an increase in Noxa and Bax gene expression levels, whereas the expression level of Survivin decreased. Similarly, Noxa gene was overexpressed in treated MCF7 cells. In treated SKBR3 cells, a decline in the c-Flip mRNA level was determined. Furthermore, release of cytokines, IL-6, IL-10, and TNF-alpha, was determined in cell culture supernatants. CpG/DcMNP treatment leads to an increase in the release of IL-6 in MDA-MB231 and SKBR3 cells, whereas release of IL-10 and TNF-alpha did not change significantly. It is indicated that CpG-ODN may show its cytotoxic effect by regulating the expression of apoptosis-related genes and the release of cytokine in breast cancer cells. (C) 2016 Elsevier B.V. All rights reserved.

It has been reported that the Natural killer (NK) cell with FCGR3A (CD16a) V genotype is associated with enhanced clinical response to IgG1 monoclonal Ab (mAb) therapy such as trastuzumab, rituximab and cetuximab (1,2), suggesting a role of antibody-dependent cell-mediated cytotoxicity (ADCC) induced by NK cells. NK cells express three types of polymorphism of CD16; FcγRIIIa-158 VV, VF, FF, which are derived from the genotype of FCGR3A. It is a clinical challenge to improve the outcome in patients with FCGR3A 158FF genotype whose NK cells have lower affinity to mAb and mediate poor ADCC activity. The IL-15 superagonist/IL-15Rα-Fc fusion complex (ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, inducing their expansion, cytotoxity, and ADCC against B cell lymphoma (3, 4, 5). Here, we examined the effect of ALT803 on NK cell-mediated ADCC activity by the the anti-HER2 IgG1 mAb trastuzumab in HER2+ cell lines (SKBR3, BT474, MDA-MB-453). In addition, we used the anti-epidermal growth factor receptor(EGFR) IgG1 mAb cetuximab in EGFR positive TNBC cell lines (MDA-MB-231, SUM149, BT549). Finally, we examined the anti-PD-L1 IgG1 mAb avelumab was used for PD-L1 positive breast cancer cell lines (MDA-MB-231, BT549). Trastuzumab, cetuximab, and avelumab all significantly increased NK cell-induced lysis via ADCC. ALT803 significantly further increased both NK induced lysis and ADCC activity in all the cell lines. There was a significant positive correlation for the mean of ADCC lysis induced by NK cells from three FF (21%), three VF (33%), three VV (45%) donors. ALT803 significantly increased the mean of ADCC lysis by NK cells from all donors of each genotype to the same extent. ALT803 increased the expression of NK cell-activating receptors and cytotoxic granules regardless of the genotype of NK cell FCGR3A in terms VV, VF, or FF. We further examined the potential of ALT803 for NK cell-cytotoxicity suppressed by TGF-β1 which is one of the main barriers to immunity in the tumor microenvionment (TME). NK cells treated with TGF-β1 showed lower expression of activating receptors and cytotoxic granules, culminating in decreased lysis of MDA-MB231. ALT803 inhibited TGF-β1 from down-regulating the expression of NK cell-activating receptors and cytotoxic granules, and from suppressing the cytotoxicity of NK cells to MDA-MB231. In conclusion, the IL-15 superagonist ALT803 can potentially increase the clinical benefit of ADCC-based mAb therapy for breast cancer patients, regardless of the genotype of FCGR3A. Moreover, ALT803 prevented NK cell-cytotoxity from TGF-β1-induced suppression, providing a rationale for ALT803 therapy to overcome TME-mediated immunosuppression. References (1) Gavin et. al. JAMA Oncol.2017;3(3) (2) Musolino et. al. J Clin Oncol.2008;26(33) (3) Xu et. al. Cancer Res.2013;73(10) (4) Kim et. al. Oncotarget.2016;7(13) (5) Rosario et. al. Clin. Cancer Res. 2016; 22(3) Citation Format: Fujii R, Wong HC, Schlom J, Hodge JW. An IL-15 superagonist enhances antibody-dependent cell-mediated cytotoxicity against breast cancer cells regardless of FCGR3A (CD16) genotype and rescues NK cell from TGF-β1-induced immunosuppression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-04.

Background: The miR-17-92 is an oncogenic miRNA cluster that generate six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1. Accumulating evidences indicate the oncogenic role of the miR-17-92 cluster in human cancers. Amplification of 13q31-q32, which is the locus of the miR-17-92 cluster, have been reported in haematopoietic malignancies, such as B cell lymphoma. In contrast, MIR17HG was deleted in 21.9% of breast cancers and loss of heterozygosity at 13q12-q13 was associated with poor prognosis in breast cancer. This oncogenic miRNA cluster is deferentially expressed in various cancer. Since, miR-17-92 cluster shows differential expression among the cancers types, it is hypothesized that biological function may also vary depending on the context. Indeed, this cluster has been shown to act a tumour suppressor in some cancers. However, the functional role of this cluster in the subtypes of breast cancer remains largely unknown. Methods: The oncomine datasets were analysed for expression of MIR17HG in breast cancer at parameters p-value threshold of 0.01 with minimum 2-fold change. We generated stable sub-clones of MCF7, T47D, SKBR3 and MDA-MB231 cells overexpressing miR-17-92 cluster by transducing with lentivirus expressing miR-17-92. Proliferation was assessed by MTS assay and colony forming assay. Cell migration was tested using scratch method. Invasion potentiality was monitored by using matrigel Boyden chamber invasion assay. For drug response analysis, control and microRNA overexpressing sub-clones were exposed to different chemotherapeutic agents at different concentrations followed by MTS assay at different time intervals. Association of miRNAs belonging to miR-17-92 with outcome in breast cancer was determined by Kaplan-Meier analysis on METABRIC dataset. Results: We observed that expression of MIR17HG was increased in tissues and cell lines from triple negative breast cancer (TNBC) but decreased in the tissues and cell lines from the estrogen receptor (ER)-positive breast cancer. Our results showed that ectopic expression of miR-17-92 cluster significantly suppressed cell proliferation, colony formation, migration and invasion of ER-positive (MCF7, T47D) and HER2-enriched (SKBR3) cells whereas it enhanced cell proliferation, colony formation, migration and invasion in (TNBC) MDA-MB 231 cells. Further, we found that expression of miR-17-92 cluster sensitized MCF7 cells whereas it rendered SKBR3 cells resistant to chemotherapeutic compounds. Higher expression of five miRNAs of this cluster was associated with better relapse free survival (RFS) in (miR-17, miR-19a, miR-20a, miR-19b and miR-92) Luminal A subtype whereas three miRNAs of this cluster were associated with poor RFS in (miR-17, miR-18a and miR-92) HER2-enriched and (miR-17, miR-19b and miR-92) TNBC subtypes. Conclusions: Taken together our results suggest that miR-17-92 cluster acts as a tumour suppressor in ER-positive and HER2-enriched breast cancer cells but shows oncogenic role in TNBC. Our observations underscore the functional complexity of miR-17-92 in a context-dependent and cell type-dependent manner, and more investigations are warranted to fully explore the functional complexity of miR-17-92 in subtypes of breast cancer. Citation Format: Hossain MM, Arabkari V, Barua D, Gupta A, Islam MN, Gupta S. miR-17-92 cluster, an oncogenic microRNA cluster acts as a context dependent tumour suppressor in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-14.

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

Purpose Targeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed. Methods To identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN+/− spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor. Results Treatment of tumor-bearing MMTV-NIC-PTEN+/− mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib. Conclusion Together these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.

Mammospheres are breast cancer stem cells (BCSCs) that could be yielded through culturing cells in non-adherent and non-differentiating condition. With regard to therapy resistance of cancer stem cells (CSCs), it is essential to discover efficient approaches targeting CSCs. Viola odorata extract has been considered as a traditional herbal anti-metastatic drug in several cancer cells. Effect of this drug on BCSCs has not been clearly identified. Current study tries to detect and to compare effect of Viola odorata extract on malignant characterization of breast cancer cell lines and BCSCs. MCF7 and SKBR3 and their derived mammospheres as BCSCs were used and the effect of alcoholic extraction of Viola odorata on apoptosis and malignant characters of MCF7, SKBR3 and their derived BCSCs were analyzed and compared. Viola odorata extract induced cell death in MCF7, SKBR3 and their derived mammospheres through apoptosis without any effects on MCF10A. Also, this extract showed anti-migratory, anti-invasion and anti-colony formation activity in MCF7, SKBR3 and their derived mammospheres which was significantly more in MCF7- and SKBR3-derived mammospheres. Also, this extract decreased size and volume of tumors generated by MCF7, SKBR3 and their derived mammospheres in chicken embryo model. Viola odorata extract exerted anti-cancerous activity on both breast cancer cell lines and their derived BCSCs. Anti-cancerous activity of this extract was significantly more in MCF7-, SKBR3-derived mammospheres in comparison with dedicated cell lines. Data suggest that Viola odorata extract mostly targets cancerous cells, not normal cells with exception in high concentration. It acts in a cell-dependent manner.

Exosomes are nano-sized vesicles, which are currently under intensive study as potential diagnostic biomarkers for many health disorders, including cancer. This paper addresses the study of an electrochemical immunosensor in different formats for the characterization and quantification of exosomes derived from three breast cancer cell lines (MCF7, MDA-MB-231 and SKBR3). To achieve that, the exosomes were preconcentrated from cell-culture supernatant (and eventually in human serum) on magnetic particles modified with antibodies against the general tetraspanins CD9, CD63 and CD81, as well as specific receptors of cancer (CD24, CD44, CD54, CD326 and CD340). The electrochemical immunosensor is able to reach a limit of detection of 105 exosomes μL-1 directly in human serum, when performing the immunomagnetic separation with antiCD81 modified magnetic particles and the labeling based on CD24 and CD340 as cancer-related biomarker, avoiding the interference from free receptors in the serum matrix. Furthermore, the electrochemical immunosensor shows reliable results for the differentiation of healthy donors and breast cancer individuals based on specific epithelial biomarkers. This approach is a highly suitable alternative method for the detection of exosomes in scarce resource settings.

Background: Apoptosis is a crucial process in the failure of cancer progression. However, the occurrence of resistance to chemotherapy in cancer cells may prevent apoptosis via induction of the expression of the anti-apoptotic genes (Bcl-2) and/or reduction of the expression of the apoptotic caspases. Objectives: The current study aimed at investigating the apoptotic effects of targeted co-delivery of docetaxel and cMet siRNA (siMet) through mucin1 aptamer-conjugated chitosan nanoparticles on mucin1 + metastatic breast cancer cells (SKBR3). Methods: Characterization of nano-drugs,Western blotting assay, annexin V/ propidium iodide staining assay, and gene expression studies were evaluated based on metastatic breast cancer cells. Results: Characterization showed the appropriate size (110.5 3.9 nm), zeta potential (11.6 0.8 mV), and spherical shape of nanoparticles. Loading efficiency of 90.7% and 88.3% were gained for siRNA and docetaxel, respectively. The siRNA entrapment onto nanoparticles and conjugation of aptamers to nanoparticles were confirmed by gel electrophoresis. Gene knockdown assay represented the effectiveness of siMet on cMet gene silencing. According to the flow cytometry results, targeted co-delivery was successful in leading tumor cells to apoptosis (94.9%). Also, targeted co-delivery could reduce the expression of Bcl-2 gene (P < 0.0001) and increase the expression of caspase-8 and caspase-9 genes (P < 0.0001). Conclusions: Combination treatment of metastatic breast cancer cells with aptamer-conjugated nanoparticles containing docetaxel and cMet siRNA significantly increased apoptosis.

Many dietary flavonoids possess anti-cancer activities. Here, the effect of apple peel flavonoid fraction 4 (AF4) on the growth of triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2-positive (SKBR3) breast cancer cells was determined and compared with the effect of AF4 on normal mammary epithelial cells and dermal fibroblasts. AF4 inhibited breast cancer cell growth in monolayer cultures, as well as the growth of MCF-7 spheroids, without substantially affecting the viability of non-malignant cells. A sub-cytotoxic concentration of AF4 suppressed the proliferation of MDA-MB-231 cells by inhibiting passage through the G0/G1 phase of the cell cycle. AF4-treated MDA-MB-231 cells also exhibited reduced in vitro migration and invasion, and decreased Akt (protein kinase B) signaling. Higher concentrations of AF4 were selectively cytotoxic for MDA-MB-231 cells. AF4 cytotoxicity was associated with the intracellular accumulation of reactive oxygen species. Importantly, intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice. The selective cytotoxicity of AF4 for breast cancer cells, combined with the capacity of sub-cytotoxic AF4 to inhibit breast cancer cell proliferation, migration, and invasion suggests that flavonoid-rich AF4 (and its constituents) has potential as a natural therapeutic agent for breast cancer treatment.

The exosomes are emerging as biomarkers for the detection of cancer in early stages, as well as for the follow-up of the patients under treatment. This paper describes the characterization of exosomes derived from three different breast cancer cell lines (MCF7, MDA-MB-231 and SKBR3), and the quantification based on a magneto-actuated immunoassay. The exosomes are separated and preconcentrated on magnetic particles by immunomagnetic separation and labelled with a second antibody conjugated with an enzyme for the optical readout performed with a standard microplate reader. Several molecular biomarkers, including the general tetraspanin CD9, CD63 and CD81, and the receptors related with cancer (CD24, CD44, CD54, CD326 and CD340) were studied either for the immunomagnetic separation or the labelling, in different formats. After a rational selection of the biomarkers, this immunoassay is able to detect 105 exosomes μL-1 directly in human serum without any treatment, such as ultracentrifugation. The interference from free receptors in the samples could easily be prevented by performing the immunomagnetic separation with antiCD81 modified magnetic particles and the labeling based on either CD24 or CD340. Furthermore, the differentiation of healthy donors and breast cancer individuals was also demonstrated. This approach is a highly suitable alternative method for flow cytometry, providing a sensitive method for the multiplex detection but using instrumentation widely available in resource-constrained laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.

Breast cancer is the second cause of death in the world. Ionising radiation is a potent mutagen that can cause DNA damage, chromosomes breakage, and cell death. In the present study, radiotherapy and nanoparticle-antibodies (ABs) have been combined to enhance the efficacy of cancer cell treatment. Silver nanoparticles (SNP) were synthesised, coated with anti-HER2, and then characterised with different techniques such as X-ray diffraction, dynamic light scattering, transmission electron microscopy, Fourier transform infrared, and UV-Vis spectroscopy. SKBR3 cells were irradiated with cobalt-60 in the presence of nanoparticle-AB as the drug. Cell viability was measured using the diphenyltetrazolium bromide assay, and the cellular status was assessed by Raman spectroscopy. Irradiation considerably decreased cell viability proportionate to the dose increase and post-irradiation time. The surface-enhanced Raman spectroscopy increased the signal in the presence of SNP. Increasing the dose to 2 Gy increased the irradiation resistance, and higher dose increases (4 and 6 Gy) enhanced the irradiation sensitivity. Moreover, the cellular changes induced by irradiation in the presence of the drug were stable after 48 h. The authors results introduced the combination of the drug with radiation as an effective treatment for cancer and Raman spectroscopy as a suitable tool to diagnose effective irradiation doses.

Preussin, a hydroxyl pyrrolidine derivative isolated from the marine sponge-associated fungus Aspergillus candidus KUFA 0062, displayed anticancer effects in some cancer cell lines, including MCF7. Preussin was investigated for its cytotoxic and antiproliferative effects in breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231), representatives of major breast cancers subtypes, and in a non-tumor cell line (MCF12A). Preussin was first tested in 2D (monolayer), and then in 3D (multicellular aggregates), cultures, using a multi-endpoint approach for cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), resazurin and lactate dehydrogenase (LDH)) and proliferative (5-bromo-2&prime, deoxyuridine (BrdU)) assays, as well as the analysis of cell morphology by optical/electron microscopy and immunocytochemistry for caspase-3 and ki67. Preussin affected cell viability and proliferation in 2D and 3D cultures in all cell lines tested. The results in the 3D culture showed the same tendency as in the 2D culture, however, cells in the 3D culture were less responsive. The effects were observed at different concentrations of preussin, depending on the cell line and assay method. Morphological study of preussin-exposed cells revealed cell death, which was confirmed by caspase-3 immunostaining. In view of the data, we recommend a multi-endpoint approach, including histological evaluation, in future assays with the tested 3D models. Our data showed cytotoxic and antiproliferative activities of preussin in breast cancer cell lines in 2D and 3D cultures, warranting further studies for its anticancer potential.

In the present work, the in vitro anti-proliferative and anti-bacterial activities of three semi-synthetic benzoate pinocembrin derivatives, isolated from the aerial parts of Glycyrrhiza glabra L., were investigated. As occurs in most natural compounds, the bioavailability of pinocembrin is very poor, therefore it should be improved by chemical strategies aimed to prolong its shelf life and, consequently, its activity. On this basis, three benzoate derivatives of pinocembrin (a1–a3) were synthesised and assayed in order to ascertain their biological value. Among them, compound a1 showed the highest anti-proliferative activity on a wide panel of cancer cell lines, as well as low toxic effects on non-malignant breast cells. The calculated IC50 values in HeLa and SKBR3 cells were 8.5 and 12.7 µM, respectively. Briefly, a1 treatment increased ROS levels, induced mitochondrial membrane damage leading to necrotic death of HeLa cells. Moreover, a1 displayed a promising anti-bacterial activity.

Yinxing Shen,1,2 Mengya Li,1 Tianqi Liu,1 Jing Liu,2 Youhua Xie,2 Junqi Zhang,2 Shouhong Xu,1 Honglai Liu11Key Laboratory for Advanced Materials, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China; 2Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, People’s Republic of ChinaPurpose: As well as functioning as a ligand that is selectively internalized by cells overexpressing human epidermal growth factor receptor-2 (HER2), HApt can exert cytotoxic effects by inducing cross-linking and subsequent translocation of HER2 to cytoplasmic vesicles, such downregulation of HER2 inhibits cell proliferation and induces apoptosis. We aimed to exploit the potential of HApt as both a targeting agent and antagonist to maximize the efficacy of mesoporous silica nanoparticle (MSN)-based drug release systems for HER2-positive breast cancer.Materials and methods: We fabricated novel HApt aptamer-functionalized pH-sensitive β-cyclodextrin (β-CD)-capped doxorubicin (DOX)-loaded mesoporous silica nanoparticles (termed MSN-BM/CD-HApt@DOX) for targeted delivery and selective targeting of HER2-positive cells. MSN-functionalized benzimidazole (MSN-BM) was used to load and achieve pH stimuli-responsive release of the chemotherapeutic agent doxorubicin (DOX). β-cyclodextrin was introduced as a gatekeeper for encapsulated DOX and HApt as a selective HER2-targeting moiety and biotherapeutic agent.Results: Physical and chemical characterizations (FT-IR, XRD, TEM and BET) confirmed successful construction of MSN-BM/CD-HApt@DOX nanoparticles. In vitro release assays verified pH-sensitive DOX release. MSN-BM/CD-HApt@DOX (relative DOX concentration, 3.6μg/mL) underwent HER2-mediated endocytosis and was more cytotoxic to HER2-positive SKBR3 cells than HER2-negative MCF7 cells. MSN-BM/CD-HApt@DOX also exhibited better uptake and stronger growth inhibition in SKBR3 cells than the control MSN-BM/CD-NCApt@DOX functionalized with a scrambled nucleotide sequence on CD. Overall, intracellular delivery of DOX and the biotherapeutic agent HApt resulted in synergistic cytotoxic effects in HER2-positive cancer cells in comparison to either DOX or HApt alone.Conclusion: MSN-BM/CD-HApt@DOX enables HER2-mediated targeting and biotherapeutic effects as well as pH-responsive DOX drug release, resulting in synergistic cytotoxic effects in HER2-overexpressing cells in vitro. This novel nanocarrier could potentially enable specific targeting to improve the efficacy of chemotherapy for HER2-positive cancer.Keywords: mesoporous silica nanoparticle, pH-sensitive nanovalve, HER2 aptamer, synergistic cytotoxicity

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing second messenger which triggers Ca(2+) release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca(2+) release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca(2+) release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca(2+) release. In mammalian cell homogenates, incubation with low concentrations of [(32)P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [(32)P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [(32)P]5-N(3)-NAADP.

Trace amine-associated receptors are G protein-coupled receptors of which TAAR1 is the most well-studied. Recently, Vattai et al. (J Cancer Res Clin Oncol 143:1637–1647 https://doi.org/10.1007/s00432-017-2420-8 , 2017) reported that expression of TAAR1 may be a marker of breast cancer (BC) survival, with a positive correlation also suggested between TAAR1 expression and HER2 positivity. Neither a role for TAAR1 in breast tissue, nor in cancer, had previously been suspected. We, therefore, sought to provide independent validation and to further examine these putative relationships. First, a bioinformatic analysis on 58 total samples including normal breast tissue, BC-related cell lines, and tumour samples representing different BC sub-types found no clear correlation between TAAR1 mRNA levels and any BC subtype, including HER2 + . We next confirmed the bioinformatics data correlated to protein expression using a well validated anti-human TAAR1 antibody. TAAR1 mRNA levels correlated with the relative intensity of immunofluorescence staining in six BC cell lines (MCF-7, T47D, MDA-MB-231, SKBR3, MDA-MB-468, BT-474), but not in the MCF-10A immortalized mammary gland line, which had high mRNA but low protein levels. As expected, TAAR1 protein was intracellular in all cell lines. Surprisingly MCF-7, SKBR3, and MDA-MB-468 showed pronounced nuclear localization. The relative protein expression in MCF-7, MDA-MB-231, and MCF-10A lines was further confirmed by semi-quantitative flow cytometry. Finally, we demonstrate that the commercially available anti-TAAR1 antibody has poor selectivity, which likely explains the lack of correlation with the previous study. Therefore, while we clearly demonstrate variable expression and sub-cellular localization of TAAR1 across BC cell lines, we find no evidence for association with BC subtype.

Objective: Despite advanced treatment options available, drug resistance develops in breast cancer (BC) patients requiring novel effective drugs. Stylissa carteri, a marine sponge predominantly living in Indonesia territories, has not been extensively studied as anti-cancer. Therefore, this study targeted to assess the anti-tumor activity of the ethanol extract of S. carteri in BC cells. Methods: S. carteri was collected from Pramuka Island, at Kepulauan Seribu National Park, Jakarta, Indonesia and extracted using ethanol. Different BC cells including MDA MB 231, MDA MB 468, SKBR3, HCC-1954 and MCF-7 cells were treated with this extract for cytotoxic analysis using MTT assay. Spheroid growth assay and apoptosis assay were conducted in HCC-1954 cells. In addition, cell migration analysis and synergistic activity with doxorubicin or paclitaxel were conducted in MDA MB 231 cells. This extract was subjected also for GC-MS analysis. Results: The results show that ethanol extract of S. carteri demonstrated a cytotoxic activity in BC cells. The IC50 of this extract was lower 15 μg/ml in MDA MB 231, MDA MB 468, SKBR3, and HCC-1954 cells. Moreover, this extract inhibited spheroids growth and induced apoptosis in HCC-1954 cells. It inhibited cell migration and demonstrated a synergistic activity with doxorubicin or paclitaxel on triggering cell death in MDA MB 231 cells. Furthermore, GC-MS analysis indicated that this extract contained 1,2-Benzenediol, Dibutyl phthalate and 9,12-Octadecadienoic acid, ethyl ester. Conclusion: Our preliminary data indicate a potential anti-tumor activity of ethanol extract of S. carteri in breast cancer cells.

Extracts from the Nepenthes plant have anti-microorganism and anti-inflammation effects. However, the anticancer effect of the Nepenthes plant is rarely reported, especially for breast cancer cells. Here, we evaluate the antitumor effects of the ethyl acetate extract of Nepenthes thorellii x (ventricosa x maxima) (EANT) against breast cancer cells. Cell viability and flow cytometric analyses were used to analyze apoptosis, oxidative stress, and DNA damage. EANT exhibits a higher antiproliferation ability to two breast cancer cell lines (MCF7 and SKBR3) as compared to normal breast cells (M10). A mechanistic study demonstrates that EANT induces apoptosis in breast cancer cells with evidence of subG1 accumulation and annexin V increment. EANT also induces glutathione (GSH) depletion, resulting in dramatic accumulations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), as well as the depletion of mitochondrial membrane potential (MMP). These oxidative stresses attack DNA, respectively leading to DNA double strand breaks and oxidative DNA damage in γH2AX and 8-oxo-2′deoxyguanosine (8-oxodG) assays. Overall these findings clearly revealed that EANT induced changes were suppressed by the ROS inhibitor. In conclusion, our results have shown that the ROS-modulating natural product (EANT) has antiproliferation activity against breast cancer cells through apoptosis, oxidative stress, and DNA damage.

Xiaoyu Li,* Shujun Xia,* Wei Zhou, Ri Ji, Weiwei Zhan Ultrasound Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China *These authors contributed equally to this work Background: Multimodal contrast agents with low toxicity and targeted modification have opened up new possibilities for specific imaging of breast cancer and shown broad application prospects in biomedicine and great potential for clinical transformation. In this work, a potential multifunctional imaging agent was developed by doping Fe into hollow silica nanoparticles (HS-Fe NPs), followed by modification with specific anti-HER2 antibodies, enabling the NPs to have dual-mode ultrasound (US)–magnetic resonance (MR)-specific imaging capacity with low toxicity.Methods: Anti-HER2 antibodies were conjugated to silane–polyethylene glycol (PEG)–COOH-modified HS-Fe (HS-Fe-PEG) NPs to produce HER2-targeted HS-Fe-PEG (HS-Fe-PEG-HER2) NPs. The toxicity of HS-Fe-PEG-HER2 NPs on targeted cells invitro and blood and organ tissue of mice invivo was investigated. Distribution invivo was also studied. Confocal laser-scanning microscopy and flow cytometry were used to evaluate the targeting ability of HS-Fe-PEG-HER2 NPs invitro. US and MR instruments were used for imaging both invivo and in vitro.Results: The obtained HS-Fe-PEG-HER2 NPs (average diameter 234.42±48.76 nm) exhibited good physical properties and biosafety. In solution, they showed obvious enhancement of the US signal and negative contrast in T2-weighted MR imaging. The binding rate of HS-Fe-PEG-HER2 NPs to targeted cells (SKBR3) was 78.97%±4.41% invitro. US and MR imaging invivo confirmed that the HS-Fe-PEG-HER2 NPs were delivered passively into the tumor region of SKBR3 and bound specifically to tumor cells. Target enhancement was better than untargeted and targeted competition groups.Conclusion: HS-Fe-PEG-HER2 NPs have potential as a low-cytotoxicity and dual-mode US–MR-specific imaging agent. Keywords: dual-mode, ultrasound imaging, magnetic resonance imaging, HER2, breast cancer

In cancer treatment, polymeric nanoparticles (NPs) can serve as a vehicle for the delivery of cytotoxic proteins that have intracellular targets but that lack well-defined mechanisms for cellular internalization, such as saporin. In this work we have prepared PEGylated poly(lactic acid-co-glycolic acid-co-hydroxymethyl glycolic acid) (PLGHMGA) NPs for the selective delivery of saporin in the cytosol of HER2 positive cancer cells. This selective uptake was achieved by decorating the surface of the NPs with the 11A4 nanobody that is specific for the HER2 receptor. Confocal microscopy observations showed rapid and extensive uptake of the targeted NPs (11A4-NPs) by HER2 positive cells (SkBr3), but not by HER2 negative cells (MDA-MB-231). This selective uptake was blocked upon pre-incubation of the cells with an excess of nanobody. Non-targeted NPs (Cys-NPs) were not taken up by either type of cells. Importantly, a dose-dependent cytotoxic effect was only observed on SkBr3 cells when these were treated with saporin-loaded 11A4-NPs in combination with photochemical internalization (PCI), a technique that uses a photosensitizer and local light exposure to facilitate endosomal escape of entrapped nanocarriers and biomolecules. The combined use of saporin-loaded 11A4-NPs and PCI strongly inhibited cell proliferation and decreased cell viability through induction of apoptosis. Also the cytotoxic effect could be reduced by an excess of nanobody, reinforcing the selectivity of this system. These results suggest that the combination of the targeting nanobody on the NPs with PCI are effective means to achieve selective uptake and cytotoxicity of saporin-loaded NPs.

Targeted theranostic platform that integrates multi-modal imaging and therapeutic function is emerging as a promising strategy for earlier detection and precise treatment of cancer. Herein, we designed targeted gold-nanoshelled poly (lactic-co-glycolic acid) (PLGA) magnetic hybrid nanoparticles carrying anti-human epidermal growth factor receptor 2 (Her2) antibodies (Her2-GPH NPs) for dual-modal ultrasound (US)/magnetic resonance (MR) imaging and photothermal therapy of breast cancer. The agent was fabricated by coating gold nanoshell around PLGA nanoparticles co-loaded with perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs), followed by conjugating with anti-Her2 antibodies. Cell-targeting studies demonstrated receptor-mediated specific binding of the agent to Her2-positive human breast cancer SKBR3 cells, and its binding rate was significantly higher than that of Her2-negative cells (P

Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. Five ligands exhibited promising results with relatively low IC50 values on cells that amplify HER2 and high IC50 on those that do not express such a receptor. The most potent compound (compound 13) showed an IC50 of 0.046 µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC50 values were scored. The IC50 values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.

Aims The migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). This study aims to evaluate the biological effects of MIIP in breast cancer by targeting IGFBP2. Materials and methods Reverse transcription quantitative real-time polymerase chain reaction and Western blotting were used to evaluate the abnormal expression of MIIP and IGFBP2 in breast cancer tissue or breast cancer cell lines. Transfection assay was used to overexpress MIIP protein in breast cancer cells. MTT assay and colony formation assay were used to detect cell viability of breast cancer cells after MIIP overexpression. Transwell and wound-healing assays were used to detect cell invasion and migration after MIIP overexpression. Results MIIP was significantly decreased and IGFBP2 was significantly increased in breast cancer tissues versus para cancerous. Breast cancer tissues of HER2 overexpression and Basal-like were more significant than Luminal A and Luminal B. MIIP was obviously downregulated and IGFBP2 was upregulated in MDA-MB-231, SKBR3 and MCF-7 versus MCF-10A especially in MDA-MB-231. Cell proliferation, cell migration and cell invasion were significantly inhibited after overexpression of MIIP. IGFBP2 was downregulated after overexpression of MIIP. The effects of MIIP on cell proliferation, cell migration and invasion were significantly reversed by IGFBP2. Conclusion The abnormal expression of MIIP in breast cancer affects the cell biological effects. IGFBP2 was regulated via MIIP which may be associated with these biological effects. These results reveal that MIIP can be a potential target for breast cancer treatment.

[Image: see text] The potent N-methyl-d-aspartate (NMDA) receptor antagonists 1–3 have been demonstrated to show antiproliferative and cytotoxic effects in MCF-7 and SKBR3 breast cancer cell lines. To improve the knowledge about the role played by the NMDA receptor in the antitumor activity of these compounds, the enantiomers of 1 were prepared and evaluated for their affinity for the phencyclidine (PCP) site of the NMDA receptor and for their cytotoxic effect in MCF-7 and SKBR3 cell lines, both expressing the NMDA receptor. The (S)-1 enantiomer, showing negligible affinity for the PCP site, exhibited antiproliferative activity higher than that of (R)-1, which instead bound the PCP site. The downregulation of NMDA GluN1 expression resulted in a decreased (S)-1-induced cytotoxicity and apoptotic cell death, unequivocally demonstrating the involvement of the NMDA receptor in the antitumor effect of this compound. Due to its interesting biological profile, (S)-1 represents a lead compound to develop novel antitumor agents for breast cancer treatment.

Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter region. Methods: The MTT test was performed to determine the effective dose of TMZ. The time-dependent cell survival test was performed after the IC50 value was found. Western blotting was performed to determine MGMT gene expression levels. High Resolution Melting (HRM) technique was used to determine the methylation levels of MGMT gene promoter region. Results: TMZ has been shown to have a high cytotoxic effect on SKBR3 cell line and low cytotoxicity on MCF-7. When MGMT expression levels before and after TMZ treatment were observed by western blotting, the gene expression levels of TMZ treatment were shown to decrease in both cell lines. It was observed that MGMT gene promoter region was hypermethylated in two cell lines, and that the application of TMZ further increased the methylation levels in the promoter region. Conclusions: It was seen that TMZ could be used as a single agent in SKBR-3 cell line. With this study on breast cancer, it is expected that temozolomide treatment will lead future in vitro and in vivo studies for breast cancer.

HER2-positive breast cancer correlates with more aggressive tumor growth, a poorer prognosis and reduced overall survival. Currently, trastuzumab (Herceptin), which is an anti-HER2 antibody, is one of the key drugs. There is evidence indicating that conjugation of trastuzumab with chemotherapy drugs, such as doxorubicin (DOX), for multiple targets could be more effective. However, incomplete penetration into tumors has been noted for those conjugates. Compared to an antibody, peptides may represent an attractive alternative. For HER2, a similar potency has been observed for a 12-amino-acid anti-HER2 peptide mimetic YCDGFYACYMDV-NH2 (AHNP, disulfide-bridged) and full-length trastuzumab. Thus, a peptide, GPLGLAGDDYCDGFYACYMDV-NH2, which consists of AHNP and an MMP-2 cleavable linker GPLGLAGDD, was first designed, followed by conjugation with DOX via a glycine residue at the N-terminus to form a novel DOX-peptide conjugate MAHNP-DOX. Using HER2-positive human breast cancer cells BT474 and SKBR3 as in vitro model systems and nude mice with BT474 xenografts as an in vivo model, this conjugate was comprehensively characterized, and its efficacy was evaluated and compared with that of free DOX. As a result, MAHNP-DOX demonstrated a much lower in vitro IC50, and its in vivo extent of inhibition in mice was more evident. During this process, enzymatic cleavage of MAHNP-DOX is critical for its activation and cellular uptake. In addition, a synergistic response was observed after the combination of DOX and AHNP. This effect was probably due to the involvement of AHNP in the PI3K–AKT signaling pathway, which can be largely activated by DOX and leads to anti-apoptotic signals.

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron-dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA-PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA-PpIX fluorescence in tumor cells with lower FECH activity (MDA-MB-231, Hs 578T) than in tumor cells with higher FECH activity (MDA-MB-453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.

The development of theranostic platforms combining surface-enhanced Raman spectroscopy (SERS) imaging with NIR-stimulated photothermal therapy (PTT) is of utmost importance for the precise diagnosis and selective treatment of cancers, especially in superficial solid tumors. For this purpose, a versatile theranostic nanoprobe of liposomal layer-coated Au nanocages (AuNCs) was decorated with an anti-MUC18 single-chain antibody (scFv). 4-mercapto benzoic acid (p-MBA)-labeled AuNCs (p-AuNCs) were coated by a liposomal layer (p-AuNCs@lip), followed by conjugating anti-MUC18 scFv via post-insertion method to form immuno-liposomal layer-coated AuNCs (p-AuNCs@scFv-lip). Physicochemical characterizations of the p-AuNCs@scFv-lip were investigated by transmission electron microscopy (TEM) and UV-vis and Raman spectroscopy. Furthermore, the targeting ability and theranostic efficiency of the nanoprobe were evaluated for specific diagnosis and treatment of cancerous melanoma cells by flow cytometry, SERS mapping, and live/dead assay. The formation of lipid layer on p-AuNCs surface was confirmed by TEM imaging. After decorating the liposomal layer with scFv, a relevant red shift was observed in the UV-vis spectrum. Moreover, p-AuNCs@lip presented characteristic peaks in the Raman spectrum, which exhibited only a minor change after scFv conjugation (p-AuNCs@scFv-lip). Interestingly, the cellular uptake of AuNCs@scFv-lip by A375 cell line (MUC18+) showed a 24-fold enhancement compared with SKBR3 cells (MUC18-). AuNCs@scFv-lip specifically identified A375 cells from SKBR cells via SERS mapping and effectively killed A375 cells through the PTT mechanism. Taken together, this theranostic platform can provide a promising tool for both in situ diagnosis and remote-controlled thermal ablation of cancer cells.