Non-tumorigenic Breast Epithelial Cell Line Research Articles

Biological Activity, Apoptotic Induction and Cell Cycle Arrest of New Hydrazonoyl Halides Derivatives.

The hydrazonoyl halides are presently an important target in the field of medicinal chemistry. The interest in the chemistry of hydrazonoyl halides is a consequence of the fact that they undergo a wide variety of reactions which provide routes to a myriad of both heterocyclic and acyclic compounds. In addition, they have diverse biological activities such as antiviral, anthelmintic, antiarthropodal, fungicidal, herbicidal, insecticidal, pesticidal, acaricidal and miticidal Activity correlated to the presence of hydrazonoyl halides. Moreover, many applications in both industrial and pharmaceutical fields have been found to be associated with these halides. Depending on the above facts and continuation to our work, we herein report on the evaluation of the anticancer activity of these two halides prepared according to the published work and trying to know their molecular mechanism that they proceed to stop proliferation and metastasis of tumor cells by molecular tools such as real time PCR using different apoptotic genes, and cell cycle assay. The goal of this present study is to bring attention to the biological activities of hydrazonoyl halides and the molecular pathway they follow to exert their role in apoptotic death of cancer cell. Synthesis of hydrazonoyl halides 2c and 2f. The cytotoxic effect against different human cancer cell lines PC3, HepG-2, HCT-116, MCF-7 and also on normal human cell lines as MCF-10 and MCF-12 in a monolayer culture model was evaluated. Their mechanism of action inside cancer cell was evaluated using different molecular tools. Strong and promising chemotherapeutic hydrazonoyl halides (2a-2f) were evaluated for their different biological activities. As antimicrobial agents, results indicated that three compounds 2a, 2e and 2f exhibited high activity against two tested gram positive bacteria Staphylococcus aureus, Bacillus subtilis, and gram negative ones Escherichia coli, and Pseudomonas aeruginosa, the rest of the compounds were found to be moderately active against the tested microorganisms. Regarding their antifungal effect, compound 2c exhibited potent and promising effect against Candida albicans, while 2b was the most potent toward Aspergillus flavus Link. The compound 2f has repellent effect. With respect to the in vitro antitumor screening, this was done on different human cancer cell lines; namely PC3, HepG-2, HCT-116, MCF-7 and also on normal human cell lines; as MCF-10 and MCF-12 (normal breast epithelial cell and non-tumorigenic breast epithelial cell line) in a monolayer culture model where screening has been conducted at 100μg/ml (single dose test). Single dose test (100μg/ml) showed that, in case of PC3, all compounds have cytotoxic activity over 90% inhibition, 4 compounds have cytotoxic activity with 100% inhibition with Human colon cancer cell line, 4 compounds showed over 90% inhibition with MCF7 cell line and 4 compounds showed cytotoxic activity over 90% inhibition with HepG-2. Results of IC50 values for most promising compounds showed compounds with values lower than 20μM for all tested human cancer cell line. The promising hydrazonoyl halide 2c and 2f were selected for molecular study to know how they could act inside cancer cell causing death. Two biochemical tests were performed using the two halides 2c and 2f to predict their mechanism of action against breast carcinoma. Real time PCR analysis indicates that the two compounds induced the apoptosis of MCF7 cells through the up regulation of caspase-3, BAX mediated P53 mechanism but unfortunately, they promote the expression of anti-apoptotic protein BCL2. Also, cell cycle assay was performed using two different cell lines MCF7 and HCT116 and data revealed that the two compounds 2c and 2f induced apoptotic cells death of both lines via cell growth arrest at G2/M phase. In addition, it was noted that 2c induced arrest in the two lines more efficiently than 2f at G2/M phase.

Cytotoxic Activity Evaluation on Breast Cells of Guest-host Complexes Containing Artemisinin

Artemisinin is a sesquiterpene lactone with vastly proved anti-cancer effects and a low toxicity profile. However, the compound has poor water solubility, bioavailability and a short half-life. As such, the present paper aims to evaluate the cytotoxic effect on breast cells of three guest-host inclusion complexes containing artemisinin as the active compound and different cyclodextrins as hosts. These were tested using two different concentrations (i.e. 12.5 mM and 25 mM) and three cell lines, namely two human breast adenocarcinoma cell lines (MCF7 and MDA-MB-231) and one human non-tumorigenic breast epithelial cell line (MCF10A) employing the colorimetric microculture tetrazolium assay. After a 72h stimulation period, the most promising results were obtained for the complex containing artemisinin and Heptakis(2,3,6-tri-O-methyl)-b-cyclodextrin, the cell viability decrease being significant for the estrogen positive MCF7 cell line (80.0 � 2.3 %), making the complex a potential candidate for further in vivo testing.

A simultaneously quantitative profiling method for 40 endogenous amino acids and derivatives in cell lines using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Endogenous metabolites of amino acids and their derivatives in biosamples are frequently highlighted as the most differential metabolites in recent metabolomics studies. The method for the detection of amino acid derivatives such as N-acetyl amino acids and oligopeptides is rarely reported. We developed a rapid, high-throughput, sensitive and reliable quantitative method to simultaneously profile 40 underivatized amino acids and their derivatives including N-acetyl amino acids and oligopeptides in cell lines, based on ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC- MS/MS) by using a hydrophilic interaction liquid chromatography (HILIC) column. The optimized method was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability for all analytes. Only one compound (cysteine-glutathione disulfide) showed relatively low recoveries at three concentration levels (60.8–74.3%). The limit of quantification (LOQ) for most compounds was in the range of 0.6–10 ng/mL (6–100 pg on column). This method was successfully applied to the analysis of amino acids and their derivatives in breast cancer cell samples. Principal component analysis (PCA) and the orthogonal projections to latent structures (OPLS) showed a clear discrimination of the non-tumorigenic breast epithelial cell line MCF-10A from the breast cancer cell line HCC 1806. Characteristic metabolic changes in amino acid metabolism were observed in the breast cancer cell line. This quantified analytical method of 40 endogenous amino acids and their derivatives in cell lines meets the requirement of quantification in specific expanded metabolomics studies with good sensitivity.

Abstract 4605: Exosomes harvested from patient-derived-explants or cells-enhance proliferation and migration in TNBC breast cancer cells and breast epithelial cells

Abstract Triple negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype due in part by high rates of metastasis and poor prognoses. TNBC tumors are molecularly heterogeneous and lack targetable receptors common in other breast cancer subtypes (estrogen receptor, HER2/Neu amplification), hindering the discovery of effective targeted therapies. Our group has designed a series of experiments to gain insight into the role of the tumor microenvironment in TNBC proliferation, migration and invasion. Patient-derived xenografts (PDXs) are translational models that are necessary to use in the laboratory setting when studying the complex tumor microenvironment of TNBC tumors. Recent breast cancer-focused studies show exosomes can be local and systemic cell-to-cell transporters of oncogenic information. Here, we aim to better understand how tumor-derived exosomes harvested from TNBC PDX models affect cell proliferation and migration when cultured with either triple negative breast cancer cell line MDA-MB-231 or non-tumorigenic breast epithelial cell line MCF-10A. PDX tumors or cells were plated in 2D culture conditions, and exosomes were isolated and purified from the cell culture media. Proliferation and migration assays were then performed after co-culture of the exosomes (or RPMI-1640 media as a negative control) with breast cell lines (MDA-MB-231, MCF-10A). The Alamar blue dye exclusion assay was used to measure proliferation, and absorbance was measured to quantify relative proliferation amongst the different treatment groups. To evaluate cell migration, a transwell migration assay was utilized where the breast cancer cell lines were plated with either the exosomal media or standard growth media as the chemoattractant for 48 hours. For invasion experiments, the transwell migration assay was employed. Our data indicate that TNBC PDX-derived exosomes increased cell proliferation in the MDA-MB-231 cell line by an average of approximately 60% compared to the control (p ≤ 0.05). We observed similar patterns of increased cell proliferation in the MCF-10A cell line, with an increase of 34% when cells were exposed to exosomes (p ≤ 0.05) compared to the control. Exosomes from TNBC PDX models increased migration in MDA-MB-231 cells by 4% and in MCF-10A cells by 10 percent. Interestingly, when we screened the exosomes from different TNBC PDX models, we found that one invasive TNBC PDX model contributed to the increased proliferation and migration in both the MDA-MB-231 and MCF-10A cell lines. Exosomes harvested and purified from TNBC patient-derived explants, and 2D culture conditions models increased proliferation and migration, cell line MDA-MB-231, and the non-tumorigenic breast epithelial cell line MCF-10A. Our data suggests that exosomes are key mediators in the progression of cancer. Note: This abstract was not presented at the meeting. Citation Format: Jordon Coward, Margarite D. Matossian, Paige A. Roberts, Jasmine Johnson, Matthew E. Burow, KiTani P. Lemieux. Exosomes harvested from patient-derived-explants or cells-enhance proliferation and migration in TNBC breast cancer cells and breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4605.

Abstract 4281: LT-IIc, a bacterial type II heat-labile enterotoxin, induces specific lethality in triple negative breast cancer cells by modulaton of autophagy and induction of apoptosis and necroptosis

Abstract Despite the recent advances in breast cancer treatment, triple negative breast cancer (TNBC) remains a serious health problem with limited treatment options. Poor prognosis is due to the development of chemoresistance. To discover novel therapeutic approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for their capacity to induce cell death in TNBC cells. LT-IIc, but not the other toxins, significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effects on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ breast cancer cells, or SKBR-3, HER2+ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as observed by the accumulation of LC3B-II and p62 proteins. Morphologically, LT-IIc induced accumulation of enlarged LAMP2+ autolysosomes selectively in TNBC cells. Bafilomycin A1, an inhibitor of autophagic flux, blocked the formation and/or retention of these autolysosomes. The increase in caspase 3, 7 activity and annexin V staining indicated that LT-IIc induced an apoptotic response. Co-treatment with necrostatin-1, however, demonstrated that the lethal response to LT-IIc is elicited, at least in part, by concomitant induction of necroptosis. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy while simultaneously blocking autolysosomal progression in TNBC cells, resulting in specific cytotoxicity in this breast cancer subtype. Citation Format: Patricia A. Masso-Welch, Sofia Girald Berlingeri, Natalie D. King-Lyons, Lorrie Mandell, John C. Hu, Christopher Greene, Matthew Federowicz, Peter Cao, Yasser Heakal. LT-IIc, a bacterial type II heat-labile enterotoxin, induces specific lethality in triple negative breast cancer cells by modulaton of autophagy and induction of apoptosis and necroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4281.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A.Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3’,4,5’-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 μM and inhibited migration with 37.5 ± 14.8% at 3 μM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 μM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 μM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 μM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 μM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells.TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.

Exosomes Promote a Metastatic Mircroenvironment in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is the most aggressive subtype and is characterized by its patterns of metastasis and abnormal morphology. Due to the molecular heterogeneity in TNBC's, discovering effective targeted therapies has proven to be difficult. Our research group has designed a series of experiments to gain insight on how role of the microenvironment plays a role in TNBC proliferation, migration and invasion. Studies indicate that cellular stress causes exosomes to be released from both tumorigenic and nontumorigenic cells. Given that exosomes are systemic cell‐to‐cell transporters, our research goal is to better understand the role of exosomes harvested from non‐tumorigenic breast epithelial cells have on TNBC cells by measuring the proliferation, migration, and invasion when TNBC cells are exposed to these exosomes. Exosomes were harvested and purified from MCF‐10A cells. Proliferation was measured by plating 5 × 105 MDA‐MB‐231 cells in a 96 well plate which were exposed to either exosomes from MCF‐10A cells or growth media. Absorbance was measured at 560nm after 48 hours. Migration was measured by plating 5 × 10 MDA‐MD‐231 cells with the exosomal media or growth media for 48 hours. Invasion was measured using the Matrigel assay at a density of 5 × 10 5 MDA‐MB‐23 cells were exposed to exosomes from MCF10A cells. The migration and invasion assays were measured by counting cells at 4x magnification. Our data indicate that MCF‐10A exosomes increased the growth of TNBC MDA‐MB231 cells by an average of approximately 30% compared to the control (p>0.05). The MDA‐MB‐231 cells demonstrated enhanced migration and invasion when exposed to exosomes from MCF‐10A cells at 10% and 4%, respectively (p> 0.05) when compared to the control. Exosomes from nontumorigenic breast epithelial cell line MCF‐10A cells caused increases in proliferation, migration, and invasion in TNBC cell line MDA‐MB‐231. Our data suggests that exosomes are key mediators in the progression of cancer.Support or Funding InformationD34HP00006‐31‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LT-IIc, A Bacterial Type II Heat-Labile Enterotoxin, Induces Specific Lethality in Triple Negative Breast Cancer Cells by Modulation of Autophagy and Induction of Apoptosis and Necroptosis.

Triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. To discover novel approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for cytotoxicity in TNBC cells. Only LT-IIc significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effect on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ cells, or SKBR-3 cells, HER2+ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as seen by accumulation of LC3B-II and p62. Morphologically, LT-IIc induced the formation of enlarged LAMP2+ autolysosomes, which was blocked by co-treatment with bafilomycin A1. LT-IIc induced apoptosis as demonstrated by the increase in caspase 3/7 activity and Annexin V staining. Co-treatment with necrostatin-1, however, demonstrated that the lethal response of LT-IIc is elicited, in part, by concomitant induction of necroptosis. Knockdown of ATG-5 failed to rescue LT-IIc-induced cytotoxicity, suggesting LT-IIc can exert its cytotoxic effects downstream or independently of autophagophore initiation. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy, while simultaneously blocking autolysosomal progression in TNBC cells, inducing a specific cytotoxicity in this breast cancer subtype.

Design and synthesis of 2-phenyl benzimidazole derivatives as VEGFR-2 inhibitors with anti-breast cancer activity.

Three new series of 2-phenyl benzimidazole-based derivatives were designed, synthesized, and evaluated for their in vitro cytotoxic activity against breast cancer (MCF-7) cell lines. Three compounds 8, 9, and 15 showed high cytotoxic activities, with IC50 values of 3.37, 6.30, and 5.84μM, respectively, while they showed comparable cytotoxicity to the standard drug doxorubicin against human normal cells, including nontumorigenic breast epithelial cell line (MCF-10F), skin fibroblast cell line (BJ), and lung fibroblast cell line (MRC-5). Six of the synthesized compounds were screened against vascular endothelial growth factor receptor 2 (VEGFR-2) where compounds 8, 9, 12, and 15 exhibited an outstanding potency in comparison with sorafenib, with IC50 values of 6.7-8.9nM. Molecular docking study assessed the good binding patterns of the most potent compounds with the reported conserved amino acids of VEGFR-2 active site.

Long-term exposure to extremely low-dose of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce non-malignant breast epithelial cell transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathway.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.

MicroRNA-498 promotes proliferation and migration by targeting the tumor suppressor PTEN in breast cancer cells.

Triple negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and high mortality rate. The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role in cell proliferation and cell migration by negatively regulating the PI3K/Akt pathway. PTEN is downregulated by microRNAs in multiple cancers. However, few microRNAs have been reported to directly target PTEN in TNBC. In this study, microRNAs predicted to target PTEN were screened by immunoblotting and luciferase reporter assays. Expression levels of microRNA-498 (miR-498) were measured by TaqMan microRNA assays. We performed clonogenic, cell cycle and scratch wound assays to examine the oncogenic role of miR-498. We demonstrated that miR-498 directly targeted the 3'untranslated region of PTEN mRNA and reduced PTEN protein levels in TNBC cells. Compared with the non-tumorigenic breast epithelial cell line MCF-10A, TNBC cell lines overexpressed miR-498. Moreover, miR-498 promoted cell proliferation and cell cycle progression in TNBC cells in a PTEN-dependent manner. Suppressing miR-498 overexpression impaired the oncogenic effects of miR-498 on cell proliferation and cell migration. This study identified a novel microRNA (miR-498) overexpressed in TNBC cells and its oncogenic role in suppressing PTEN. These results provide new insight into the downregulation of PTEN and indicate a potential therapeutic target for treating TNBC.

PO-420 Mechanisms of action of anti-proliferative lichen compound protolichesterinic acid

Introduction Protolichesterinic acid (PA), an aliphatic α-methylene γ-lactone isolated from the lichen Iceland moss ( Cetraria islandica ) has an anti-proliferative effect against a variety human cancer cells. In multiple myeloma cells PA induces apoptosis. The anti-proliferative effect of PA is not mediated by the known inhibitory action of PA on 5- and 12-lipoxygenases and effects on growth factor signalling are secondary. PA inhibits DNA replication and earlier studies indicated direct effects of PA on HIV reverse transcriptase (RT) and DNA polymerase. RT inhibitors are known to affect mitochondria. Material and methods Molecular modelling software (Glide, Schrodinger, LLC) was used to test for the ability of PA to bind onto DNA polymerases α, β, γ and θ. The crystal structures of polymerases were acquired from Protein Data Bank (PDB ID: 4QCL, 5TB8, 4ZTZ and 5A9F, respectively) and PA was docked into each structure. The effects of PA on mitochondria were assessed morphologically by electron microscopy and metabolically by measuring extracellular glucose and lactate concentrations in cell medium after 6 and 24 hour (ABL90 FLEX blood gas analyzer) in the pancreatic cancer cell line Aspc-1, the breast cancer cell line T47D, normal skin fibroblast and MCF-10 non-tumorigenic breast epithelial cell line. Results and discussions PA docked to all four types of DNA polymerases, creating multiple hydrogen bonds directly with the enzymes/polymerases and with the metal ion (Mg 2+ ) cofactor. Hydrogen bonds to the metal ions bridging the interaction to the enzyme appeared important and might be necessary/important for the activity of protolichesterinic acid and explain its mechanism of action. Morphological changes in mitochondria were observed following exposure to PA in both cancer cell lines, but these were difficult to interpret. Aspc-1 pancreatic cancer showed a significant increase in lactate production after 24 hour exposure to PA. This was not observed in the breast cell lines, T47D and MCF-10. Aspc-1 is less sensitive to the anti-proliferative effects of PA than the breast cell lines. Further metabolomics studies are in progress. Conclusion Docking study of crystal structures of DNA polymerases indicates that PA could affect activity of several types of DNA polymerase, but this remains to be tested experimentally. PA may affect mitochondrial structure but increased lactate production is associated with relative resistance to its anti-proliferative effects.

Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer.

Because of the critical role of the large neutral amino acid transporter-1 (LAT-1) in promoting tumor growth and proliferation, it is fast emerging as a highly attractive biomarker for the imaging and treatment of human malignancies, including breast cancer. While multibranched gold nanoparticles (AuNPs) have emerged as a promising modality in the photothermal therapy (PTT) of cancers, some of the key challenges limiting their clinical translation lie in the need to develop reproducible and cost-effective synthetic methods as well as the selective accumulation of sufficient AuNPs at tumor sites. In this study, we report a simple and direct seed-mediated synthesis of monodispersed multibranched AuNPs using the catechol-containing LAT-1 ligands, L- and D-dopa, to confer active cancer targeting. This route obviates the need for additional conjugation with targeting moieties such as peptides or antibodies. Nanoflower-like AuNPs (AuNF) with diameters of approximately 46, 70, and 90 nm were obtained and were found to possess excellent colloidal stability and biocompatibility. A significantly higher intracellular accumulation of the L- and D-dopa functionalized AuNFs was observed in a panel of breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, and MDA-MB-453) when compared to the nontargeting control AuNFs synthesized with dopamine and 4-ethylcatechol. Importantly, no significant difference in uptake between the targeting and nontargeting AuNFs was observed in a non-tumorigenic MCF-10A breast epithelial cell line, hence demonstrating tumor selectivity. For PTT of breast cancer, Ag+ was introduced during synthesis to obtain L-dopa functionalized nanourchin-like AuNPs (AuNUs) with strong near-infrared (NIR) absorbance. The L-dopa functionalized AuNUs mediated selective photothermal ablation of the triple negative MDA-MB-231 breast cancer cell line and sensitized the cells to the anticancer drugs cisplatin and docetaxel. This work brings forward an effective strategy for the facile preparation of cancer targeting multibranched AuNPs with potential for the in vivo PTT of breast cancer.

MUC1 aptamer-capped mesoporous silica nanoparticles for controlled drug delivery and radio-imaging applications

Mucin 1 (MUC1) is a cell surface protein overexpressed in breast cancer. Mesoporous silica nanoparticles (MSNs) loaded with safranin O, functionalized with aminopropyl groups and gated with the negatively charged MUC1 aptamer have been prepared (S1-apMUC1) for specific targeting and cargo release in tumoral versus non-tumoral cells. Confocal microscopy studies showed that the S1-apMUC1 nanoparticles were internalized in MDA-MB-231 breast cancer cells that overexpress MUC1 receptor with subsequent pore opening and cargo release. Interestingly, the MCF-10-A non-tumorigenic breast epithelial cell line that do not overexpress MUC1, showed reduced (S1-apMUC1) internalization. Negligible internalization was also found for S1-ap nanoparticles that contained a scrambled DNA sequence as gatekeeper. S2-apMUC1 nanoparticles (similar to S1-apMUC1 but loaded with doxorubicin) internalized in MDA-MB-231 cells and induced a remarkable reduction in cell viability. Moreover, S1-apMUC1 nanoparticles radio-labeled with 99mTc (S1-apMUC1-Tc) showed a remarkable tumor targeting in in vivo studies with MDA-MB-231 tumor-bearing Balb/c mice.

Pro-apoptotic effect of Δ2-TGZ in “claudin-1-low” triple-negative breast cancer cells: involvement of claudin-1

40% of triple-negative breast cancer (TNBC) do not express claudin-1, a major constituent of tight junction. Patients with these "claudin-1-low" tumors present a higher relapse incidence. A major challenge in oncology is the development of innovative therapies for such poor prognosis tumors. In this context, we study the anticancer effects of ∆2-TGZ, a compound derived from troglitazone (TGZ), on cell models of these tumors. In MDA-MB-231 and Hs578T "claudin-1-low" TNBC cells, Δ2-TGZ treatment induced claudin-1 protein expression and triggered apoptosis as measured by FACS analysis (annexin V/PI co-staining). Interestingly, in the non-tumorigenic human breast epithelial cell line MCF-10A, the basal level of claudin-1 was not modified following Δ2-TGZ treatment, which did not induce apoptosis. Furthermore, claudin-1-transfected MDA-MB-231 and Hs578T cells displayed a significant increase of cleaved PARP-1 and caspase 7, caspase 3/7 activities, and TUNEL staining. RNA interference was performed in order to inhibit Δ2-TGZ-induced claudin-1 expression in both the cells. In absence of claudin-1, a decrease of cleaved PARP-1 and caspase 7 and caspase 3/7 activities were observed in MDA-MB-231 but not in Hs578T cells. Claudin-1 overexpression and Δ2-TGZ treatment are associated to apoptosis in MDA-MB-231 and Hs578T "claudin-1-low" TNBC. Moreover, in MDA-MB-231 cells, claudin-1 is involved in the pro-apoptotic effect of Δ2-TGZ. Our results suggest that claudin-1 re-expression could be an interesting therapeutic strategy for "claudin-1-low" TNBC.

A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95–97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56μg/mL) and Trypanosoma cruzi (IC50=4.85μg/mL). This toxin also induced cytotoxicity (IC50=1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Abstract P6-01-15: Novel regulation of breast cancer cell aggressiveness by cancer testis antigen

Abstract We recently demonstrated that a vesicular endocytosis associated protein SH3GL2, attenuates spontaneous metastases of breast cancer cells by inducing a mesenchymal to epithelial differentiation and the onset of the intrinsic apoptotic pathway. The present study aims to understand the molecular mechanism behind the SH3Gl2 mediated reduction of spontaneous metastasis of the breast cancer cells. We employed a cDNA microarray analysis of the SH3GL2-overexpressing breast cancer cells exhibiting reduced pulmonary metastasis and identified a 12.1 fold downregulation of SPANXB1, a cancer-testis antigen that regulates sperm motility. A limited number of studies reported an association between increased SPANXB1 expression and progression of melanoma and hepatocellular carcinoma. Augmented SPANXB1 mRNA and protein expression was evident in primary breast tumors and its upregulation was associated with pulmonary metastasis of breast cancer cells. However, the expression pattern of SPANXB1 and its role in BCa development and progression is unknown. By immunohistochemical analysis, we detected high expression (p=0.002) of SPANXB1 in 78% (18/23) of the primary breast cancer tissues and corresponding lymph node metastases compared to the matched normal breast tissues. A couple of non-tumorigenic human breast epithelial cell lines were stably transformed with SPANXB1 to understand its effect on cellular growth and progression. The SPANXB1-transformed cells exhibited increased invasion (p=0.0001) and epithelial to mesenchymal transition accompanied by an augmented expression ratio of Vimentin/E-Cadherin, molecules regulating differentiation and metastasis. The SPANXB1-transformed cells also exhibited a markedly reduced expression of SH3GL2, implicating a SPANXB1:SH3GL2 crosstalk accompanied by an enhanced production of lactate (p=0.004). Our investigation identifies new breast cancer promoting role of a cancer testis antigen, which bears potential for biomarker and targeted therapeutic development. Citation Format: Kannan A, Wells RM, Ikebe M, Dasgupta S. Novel regulation of breast cancer cell aggressiveness by cancer testis antigen [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-15.

PIK3CA mutations and TP53 alterations cooperate to increase cancerous phenotypes and tumor heterogeneity.

The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer.

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.

Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells: The role of membrane progesterone receptors

Progesterone (P4), a steroid produced during estrous cycles and gestation for maintenance of pregnancy, also plays key roles in breast development to allow lactation post-parturition. Progestins (P4 and related steroids) are also implicated in breast cancer etiology. Hormone replacement therapy containing both estrogen and progestins increases breast cancer incidence while estrogen hormone therapy lowers breast cancer risk. P4 signaling via nuclear P4 receptors (PRs) has been extensively studied in breast cancer, however, progestin signaling via non-classical membrane bound progestin receptors (MPRs and PGRMC1) remains unclear. Moreover, P4 metabolites and synthetic progestins may bind membrane progestin receptors. We hypothesized that PR-negative breast epithelial cells express non-classical progestin receptors, which activate intracellular signaling pathways differently depending on nature of progestin. Therefore, our objectives for the current study were to determine expression of MPRs and PGRMC1 in two PR-negative non-tumorigenic breast epithelial cell lines, assess progestin-mediated signaling and biological functions. We determined five MPR isoforms and PGRMC1 were present in MCF10A cells and all progestin receptors but MPRβ in MCF12A cells. MCF10A and MCF12A cells were treated with P4, select P4 metabolites (5αP and 3αHP), medroxyprogesterone acetate (MPA), or a specific MPR-Agonist (MPR-Ag) and phosphorylation of ERK, p38, JNK, and AKT was characterized following treatment. To our knowledge this is the first report of ERK and JNK activation in MCF10A and MCF12A cells with P4, P4 metabolites, MPA, and MPR-Ag. Activation of ERK and JNK in cells treated with MPR-Ag implicates MPRs may serve as the receptors responsible for their activation. In contrast, p38 activation varied with cell type and with progestin treatment. P4 and MPA promoted AKT phosphorylation in the MCF12A cell line only whereas no activation was observed in MCF10A cells. Interestingly, cellular proliferation increased in MCF10A cells treated with MPA or 5αP, while MPR-Ag tended to slightly decrease proliferation. Collectively, our data highlights the importance of investigating the effects of synthetic progestins in breast cancer biology. Our results add to the understanding that various progestins have on breast epithelial cells and underscores the importance of considering both membrane bound receptors and progestin type in breast cancer development.