Proliferation Of Breast Cancer Cell Lines Research Articles

Pectinose induces cell cycle arrest in luminal A and triple-negative breast cancer cells by promoting autophagy through activation of the p38 MAPK signaling pathway

Breast cancer patients often have a poor prognosis largely due to lack of effective targeted therapy. It is now well established that monosaccharide enhances growth retardation and chemotherapy sensitivity in tumor cells. However, Pectinose whether has capability to restrict the proliferation of tumor cells remain unclear. Here, we report that Pectinose induced cytotoxicity is modulated by autophagy and p38 MAPK signaling pathway in breast cancer cell lines. The proliferation of cells was dramatically inhibited by Pectinose exposure in a dose-dependent manner, which was relevant to cell cycle arrest, as demonstrated by G2/M cell cycle restriction and ectopic expression of Cyclin A, Cyclin B, p21and p27. Mechanistically, we further identified that Pectinose is positively associated with autophagy and the activation of the p38 MAPK signaling in breast cancer. In contrast, 3-Ma or SB203580, the inhibitor of autophagy or p38 MAPK, reversed the efficacy of Pectinose suppressing on breast cancer cell lines proliferation and cell cycle process. Additionally, Pectinose in vivo treatment could significantly inhibit xenograft growth of breast cancer cells. Taken together, our findings were the first to reveal that Pectinose triggered cell cycle arrest by inducing autophagy through the activation of p38 MAPK signaling pathway in breast cancer cells,especially in luminal A and triple-negative breast cancer.

Effects of 5-fluorouracil, thymoquinone, and mammary stem cells' exosomes on in vitro cultured breast cancer cells.

5-fluorouracil (5-FU) is an antimetabolic agent used for treating slowly growing solid tumors like breast and ovarian carcinoma. Thymoquinone (TQ) is the main biologically active constituent of Nigella sativa, it has been found to demonstrate anticancerous effects in several preclinical studies, and this is because TQ possesses multitarget nature. Stem cells-derived exosomes are in the spotlight of research and are promising tissue regenerative and anticancer cell-derived nanovesicles. Herein, we studied the antineoplastic effects of Exosomes derived from mammary stem cells (MaSCs-Exo) on breast cancer cells, alone or combined with TQ when compared to a breast cancer chemotherapeutic agent; 5-FU. Our approach included performing viability test and measuring the expression of pro-apoptotic gene (Bax), anti-apoptotic gene (BCL-2) and angiogenic gene (VEGF) on Human MCF-7 cells (breast adenocarcinoma cells), the MCF-7 cells were cultured and incubated with medium containing 5-FU (25 μg/ml), TQ (200 μg/ml), MaSCs-Exo (100 μg protein equivalent), a combination of TQ (200 μg/ml) and MaSCs-Exo (100 μg). Our obtained results show that TQ and MaSCs-Exo each can effectively inhibit breast cancer cell line (MCF-7) proliferation and growth. Also, the results show that the combination of TQ and MaSCs-Exo had higher cytotoxic effects on MCF-7 breast cancer cells than TQ or 5-FU, alone. The present study shows a promising anticancer potential of exosomes isolated from mammary stem cells; this effect was potentiated by adding TQ with MaSCs-derived exosomes.

Phytosynthesizing gold nanoparticles: Characterization, bioactivity, and catalysis evaluation

Worldwide health problem generally due to the lack of widespread and comprehensive early detection methods, the associated poor prognosis of patients diagnosed in later stages of the disease and its increasing incidence on a global scale. Indeed, the struggle to fight cancer is one of the greatest challenges of mankind. Centella Asiatica (CA) and Andrographis Paniculata (AP) are two medicinal plant extracts used in the environmentally friendly extracellular production of metallic gold nanoparticles. The size, shape, stability and crystallinity of the synthesized gold nanoparticles were characterised and various characterization methods were used; UV–Vis spectroscopy, Fourier Transform Infrared Spectroscopy, X-ray diffraction, Zeta potential, Selected Area Electron Diffraction, Energy Dispersive X-ray Spectroscopy, High Resolution Transmission Electron Microscopy equipped with elemental mapping in order to confirm the formation of gold nanoparticles. The TEM results showed that gold nanoparticles were mostly spherical in shape. Their effects on anti-bacterial, catalytic, photocatalytic and anticancer cell lines were examined. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test was used to confirm the cytotoxic properties of both gold nanoparticles samples and the results showed that both the human lung cancer and human breast cancer cell line growth and proliferation were inhibited. Strong antibacterial activity against gram-negative bacteria and moderate antibacterial activity against gram-positive bacteria were identified. It has also been shown that gold nanoparticles have catalytic properties for reducing 4-nitrophenol to 4-aminophenol. By degrading the two dyes methyl orange and congo red in the presence of nano catalysts, the photocatalytic activity of synthesized nanoparticles has been assessed.

Characterization and efficiency of Ganoderma lucidum biomass as an antimicrobial and anticancer agent

The microconstituents of Ganoderma lucidum biomass (GLB) were evaluated, along with its antimicrobial and anticancer activities. Using gas chromatography-mass spectrometry analysis, 12-octadecadienoic acid (Z,Z)- and n-hexadecanoic acid with area% values of 21.0% and 11.0% were recognized in GLB. Uncooked biomass (UCB) and microwave-cooked (CE) biomass of G. lucidum caused a significant inhibition of human breast cancer (MCF-7) cell line proliferation in a dose-dependent manner. The inhibition of MCF-7 cell proliferation was 27.22 ± 1.64% using 16 µg/mL of CB while it was 52.29 ± 1.09% using 16 µg/mL of UCB. The cytotoxicity test recorded low IC50 (25.63 ± 0.52 µg/mL) of UCE compared to the IC50 value (49.99 ± 0.94 µg/mL) of CB. Highest antimicrobial activities were recorded via using UCE, compared to CE against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Candida albicans, and Aspergillus niger. The 9,12-octadecadienoic acid (Z,Z)- and n-hexadecanoic acid were able to induce both anticancer and antibiotic-resistant properties. A key therapeutic target enzyme for evolving this resistant pathogenic activity on MCF-7 and K. pneumonia, was accessed thoroughly in silico study. The compounds described, therefore, might provide a great potential for the development of new therapeutics such as anticancer and antimicrobial agents.

Abstract 115: Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines

Abstract Although, there are several breakthroughs in the treatment of breast cancer on the past few decades, high incidence of relapse and progression after conventional therapies is deeply concerning, indicating a great need for developing new therapeutics for breast cancer. In the present study, the anti-cancer effects of combinational therapy of Letrozole (Aromatase inhibitor) or Olaparib (PARP inhibitor) with Aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), were examined on well differentiated ER-positive and poorly differentiated triple negative breast cancer cell lines, T-47D and MDA-MB-231, respectively. In addition, the effect of combined treatment was also examined by detecting cell survival, cell cycle analysis, and alterations in signaling pathway that play a role in breast cancer progression such as β -catenin, STAT3, cyclin D1, Bax, c-PARP, wild and Mutant p53. In addition the effect of combined treatments were examined on gene expression levels of Cytochromes; CYP1A1, CYP1B1, Aurora kinases; Aurora-A and Aurora-B were determined by RT-PCR.Our data showed that combined treatments of 50 nmol Letrozole or 250 nmol Olaparib with 5 mmol, 2.5mmol Aspirin for 24 and 48 h, respectively, promoted apoptosis induction through enhanced histone release and caspase-3 activation compared to control and cells treated with each drug alone. Furthermore, apoptotic induction was associated with upregulation of Bax and downregulation of Bcl-2, cyclin D1, β-catenin, and STAT3 signaling pathways in both tested cell lines compared to control, suggesting that Aspirin may enhance the sensitivity of tested cells to Letrozole or Olaparib in both (ER) positive and triple negative breast cancer cell lines. In addition, cell cycle analysis showed a cell arrest at G0/G1 phase in both cell lines. Interestingly, in T-47D cells, combined treatment of Letrozole with Aspirin significantly upregulated wild p53 at protein level and downregulated CYP1A1, CYP1B1, Aurora-A, and Aurora-B expression at gene level compared to control, suggesting that combined treatment may inhibit breast cancer cell proliferation via down-regulation of Aurora-A and Aurora-B. In MDA-MB-231 cells, combined treatment of Olaparib with Aspirin showed a significant downregulation of Mutant p53 at protein level and CYP1A1, CYP1B1, Aurora-B and upregulation of Aurora-A at gene level compared to control, suggesting that there is a crosstalk between Olaparib/Aspirin and P53 In conclusion, combined treatment of Letrozole or Olaparib with Aspirin might be a promising approach to inhibit both ER (+) and triple negative breast cancer cell lines survival and proliferation. Citation Format: Marwa Elsayed ElKamel, Salah Sheweita, Ahmed Sultan. Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 115.

Antitumor and Antibacterial Efficacy of Gallium Nanoparticles Coated by Ellagic Acid.

Cancer is a mortality contributor worldwide, and breast cancer is the most common among women. Despite the numerous breast cancer therapeutic strategies, they either have limitations or sometimes are resisted by cancer, so new approaches are needed to tackle those restrictions. Nanotechnology offers exciting leaps in the diagnosis and treatment of cancer, especially breast cancer. The main objective of this study was to investigate the effect of the newly synthesized gallium nanoparticles coated by Ellagic acid (EA-GaNPs) on the induced mammary gland carcinogenesis in female rats and their antibacterial activities comparison with standard antibiotics (Ketoconazole (100 μg/ml) and Gentamycin (4 μg/ml)) by disc diffusion method using eight different microbial species. The antitumor efficacy of EA-GaNPs was conducted both in vitro and in in vivo. The result of antimicrobial activity of EA-Ga NPs (1 mg/1 mL) revealed moderate toxicity behavior against Gram-positive {Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) and Gram-negative pathogenic bacteria {Escherichia coli, Proteus vulgarfs) also, antifungal activity was detected against {Aspergillus terreus). In vitro study showed that EA-GaNPs inhibited human breast cancer cell line (MCF-7) proliferation with IC50 of 2.86 μg/ml. Although in vivo; the administration of EA-GaNPs to DMBA-treated rats ameliorated the hyperplastic state of mammary gland carcinogenesis induced by DMBA. Additionally, EA-GaNPs administration significantly modulated the activities of ALT and AST, as well as the levels of urea and creatinine in serum. Also, EA-GaNPs administration improved the antioxidant state by increasing Superoxide dismutase activity and GSH content, and decreasing malondialdehyde content in the mammary tissue, besides enhancing the apoptotic activity through elevating the levels of caspase-3 and decreasing the protein intensities of protein kinase B & phosphatidyl inositide 3-kinases. Furthermore, a significant decrease in serum Total iron-binding capacity accompanied by a significant increase in the level of calcium was noted. So, it can be concluded that the newly synthesized nanoparticles EA-GaNPs have an efficient antitumor activity that was manifested by reduction of the viability on the human breast cancer cell line (MCF-7) in vitro. Also, in vivo against the chemically induced mammary gland carcinogenesis in a female rat model. Histopathological findings were in harmony with biochemical and molecular results showing the effectiveness of EA-GaNPs against mammary carcinogenesis. Therefore, EA-GaNPs could be a promising, potent anti-cancer compound.

Anemoside A3 activates TLR4-dependent M1-phenotype macrophage polarization to represses breast tumor growth and angiogenesis

The polarization of macrophages has been previously demonstrated to be closely related to immune and inflammatory processes in the tumorigenesis and progression of breast cancer. In the present study, Anemoside A3 (A3), an active compound from Pulsatilla saponins, was screened out and polarized M0 macrophages into the classically activated macrophages (M1-phenotype). We found that A3 is an activator of TLR4/NF-κB/MAPK signaling pathway. A3 increased the expression of CD86+ (a marker of M1 macrophage) in M0 macrophage, and increased the typical M1 macrophage pro-inflammatory cytokines TNF-α, and IL-12 expression in a TLR4-dependent manner. A macrophage-cancer cell co-culture system was established to evaluate whether A3 can could switch tumor-associated macrophages (TAMs) to the M1-phenotype. In the co-culture system, A3 increased the expression of IL-12 in macrophages, whereby suppressing MCF-7 breast cancer cell line proliferation and VEGF-mediated angiogenesis. Moreover, A3 induced M1 macrophage polarization in the 4 T1 murine breast cancer model and effectively inhibited tumor growth and tumor angiogenesis. Collectively, these findings indicated that A3 induced M1 macrophages polarization to repress breast tumorigenesis via targeting the TLR4/NF-κB/MAPK signaling pathway. This study provides a rationale for utilizing traditional Chinese medicine extracts in the immunotherapy of breast cancer.

LB822 Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in breast cancer cells

A common side effect of tamoxifen therapy in estrogen receptor (ER) positive breast cancer is hair loss/thinning. Some nutraceutical ingredients known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens or they don’t necessary activate proliferation inducer ERα, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed, alone or in combination, for MCF7, T47D and BT483 breast cancer cell line proliferation (Alamar Blue and BrdU, +/- 17β-estradiol 10nM, and/or tamoxifen 2-5μM) and for ERα/β expression (qRT-PCR, western blot and immunocytochemistry).

The ERRα-VDR axis promotes calcitriol degradation and estrogen signaling in breast cancer cells, while VDR-CYP24A1-ERRα overexpression correlates with poor prognosis in patients with basal-like breast cancer.

Vitamin D is used to reduce cancer risk and improve the outcome of cancer patients, but the vitamin D receptor (VDR; also known as the calcitriol receptor) pathway needs to be functionally intact to ensure the biological effects of circulating calcitriol, the active form of vitamin D. Besides estrogen receptor alpha (ERα), estrogen‐related receptor alpha (ERRα) has also been shown to interfere with the VDR pathway, but its role in the antitumor and transactivation activity of calcitriol is completely unknown in breast cancer (BC). We observed that ERRα functionally supported the proliferation of BC cell lines and acted as a calcitriol‐induced regulator of VDR. As such, ERRα deregulated the calcitriol–VDR transcription by enhancing the expression of CYP24A1 as well as of both ERα and aromatase (CYP19A1) in calcitriol‐treated cells. ERRα knockdown limited the effect of calcitriol by reducing calcitriol‐induced G0/G1 phase cell cycle arrest and by affecting the expression of cyclin D1 and p21/Waf. The interactome analysis suggested that Peroxisome Proliferator‐Activated Receptor Gamma Coactivator 1‐α (PGC‐1α) and Proline‐, glutamic acid‐, and leucine‐rich protein 1 (PELP1) are key players in the genomic actions of the calcitriol–VDR–ERRα axis. Evaluation of patient outcomes in The Cancer Genome Atlas (TCGA) dataset showed the translational significance of the biological effects of the VDR–ERRα axis, highlighting that VDR, CYP24A1, and ERRα overexpression correlates with poor prognosis in basal‐like BC.

Abstract PS19-28: Thymoquinone and tamoxifen co-treatment synergistically inhibit proliferation, invasion and induce apoptosis in human breast cancer cell lines in vitro and in vivo

Abstract Epidemiological studies and experimental analysis indicate that dietary factors influence thedevelopment of breast cancer, suggesting the role of natural products as modifying factors againstbreast cancer. Thymoquinone (TQ) (a dietary phytochemical compound) the main active ingredientof the volatile oil of black seed (Nigella sativa) has been used to be safe when administered to awide variety of normal cells. In addition, Tamoxifen (TAM), a nonsteroidal triphenylethylene derivateand selective ER modulator, has been used as a single agent in the treatment of ER positive breastcancer.In the present study, we report the possible chemo-sensitizing effect of TQ as a promisingconventional chemotherapeutic agent, using a panel of human breast cancer cell lines, MCF-7, ZR-75-1, T-47D and BT-20 respectively in vitro and in vivo.Our data revealed that TQ, TAM or combined treatments significantly inhibited cell viability of MCF-7, T-47D, ZR-75-1 but slightly of BT-20 cell lines consistently with down-regulation of signaltransducer and activator of transcription 3 (STAT 3), mTOR, cyclin D1, oncogenic β-catenin,signaling a specific transcription factor and PPAR-gamma in a dose dependent manner. Mostimportantly, combined treatment enhanced inhibition of cell viability, DNA fragmentation andapoptosis induction through Caspase-3 activation and Bcl-2 down regulation in all tested cell linesbut this effect was limited in BT-20 cells compared to controls. Moreover, our data was supported bymicroscopic examination using electron scanning microscopy that revealed many apoptoticalterations after combined treatment compared to each TQ or TAM. Furthermore, confocalfluorescence microscopy examination revealed that only combined treatment showed a significantinhibition of filopodia formation as a sign of invasion inhibition in both BT-20 and MCF-7 humanbreast cancer cell lines. In vivo, tumor xenotransplants of human ZR-75-1 cells revealed that onlycombined treatment showed a significant regression in tumor size by 53% after 72h and enhancedE-cadherin expression as analyzed by immunohistochemistry and confocal fluorescencemicroscopy.Taken together, our findings provide strong in vitro and in vivo molecular evidences in support ofour hypothesis that Thymoquinone synchronized as chemo-preventive agent with Tamoxifen to inhibithuman breast cancer cell lines proliferation, invasion and induce apoptosis that might have a potentialimplication in breast cancer prevention and treatment. Citation Format: Islam Yahiya, Heba Helmy, Ahmed Sultan. Thymoquinone and tamoxifen co-treatment synergistically inhibit proliferation, invasion and induce apoptosis in human breast cancer cell lines in vitro and in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-28.

The high expression of NUDT5 indicates poor prognosis of breast cancer by modulating AKT / Cyclin D signaling.

NUDIX hydrolase type 5 (NUDT5) is a kind of ADP-ribose pyrophosphatase and nucleotide metabolizing enzyme in cell metabolism. Previous studies have shown NUDT5 expression affected chromosome remodeling, involved in cell adhesion, cancer stem cell maintenance and epithelial to mesenchyme transition in breast cancer cells. Nevertheless, the role of NUDT5 in breast cancer progression and prognosis has not yet been systematically studied. This study explored the association of NUDT5 with the tumor development and poor prognosis in patients with breast cancer. Our results show that the levels of NUDT5 were upregulated in breast cancer cell lines and breast tumor tissues, and the expression of NUDT5 in breast tumor tissues increased significantly when compared with adjacent non-tumorous tissues by immunohistochemical staining of tissue microarrays. Breast cancer patients with high NUDT5 expression had a worse prognosis than those with low expression of NUDT5. In addition, the knockdown of NUDT5 suppressed breast cancer cell lines proliferation, migration and invasion, and dramatically inhibited the AKT phosphorylation at Thr308 and expression of Cyclin D1. The opposite effects were observed in vitro following NUDT5 rescue. Our findings indicated that the high expression of NUDT5 is probably involved in the poor prognosis of breast cancer via the activation of the AKT / Cyclin D pathways, which could be a prognostic factor and potential target in the diagnosis and treatment of breast cancer.

11-Oxygenated Estrogens Are a Novel Class of Human Estrogens but Do not Contribute to the Circulating Estrogen Pool.

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

Breast Cancer and Microcalcifications: An Osteoimmunological Disorder?

The presence of microcalcifications in the breast microenvironment, combined with the growing evidences of the possible presence of osteoblast-like or osteoclast-like cells in the breast, suggest the existence of active processes of calcification in the breast tissue during a woman’s life. Furthermore, much evidence that osteoimmunological disorders, such as osteoarthritis, rheumatoid arthritis, or periodontitis influence the risk of developing breast cancer in women exists and vice versa. Antiresorptive drugs benefits on breast cancer incidence and progression have been reported in the past decades. More recently, biological agents targeting pro-inflammatory cytokines used against rheumatoid arthritis also demonstrated benefits against breast cancer cell lines proliferation, viability, and migratory abilities, both in vitro and in vivo in xenografted mice. Hence, it is tempting to hypothesize that breast carcinogenesis should be considered as a potential osteoimmunological disorder. In this review, we compare microenvironments and molecular characteristics in the most frequent osteoimmunological disorders with major events occurring in a woman’s breast during her lifetime. We also highlight what the use of bone anabolic drugs, antiresorptive, and biological agents targeting pro-inflammatory cytokines against breast cancer can teach us.

Synthesis, self-assembly and anticancer drug encapsulation and delivery properties of cyclodextrin-based giant amphiphiles

Cyclodextrin-calixarene giant amphiphiles that can self-assemble into nanospheres or nanovesicles have the ability to encapsulate the anticancer hydrophobic drugs docetaxel, temozolomide and combretastatin A-4 with encapsulation efficiencies >80% and deliver them to tumoral cells, enhancing their therapeutic efficacy by 1-3 orders of magnitude. These amphiphiles were modified by inserting a disulfide bridge confering them redox responsiveness. Disassembly of the resulting nanocompounds and cargo release was favored by high glutathione levels mimicking those present in the tumor microenvironment. Anticancer drug-loaded nanoformulations inhibited prostate, breast, glioblastoma, colon or cervix cancer cell lines proliferation with IC50 values markedly below those observed for the free drugs. Cell-cycle analysis indicated a similar mechanism of action for drug-loaded nanocompounds and free drugs. The results strongly suggest that the cyclodextrin-calixarene heterodimer prototype is an excellent scaffold for nanoformulations aimed to deliver anticancer drugs with limited bioavailability due to low solubility to tumoral cells, markedly increasing their effectivity.

Abstract 1962: Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors

Abstract Tucatinib is an investigational, oral, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2, without significant inhibition of EGFR. Recently, HER2CLIMB (NCT02614794), a pivotal, randomized, international, double-blind trial that evaluated tucatinib or placebo in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (MBC) with or without brain metastases, after progression with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1), showed superior progression-free and overall survival in patients treated on the tucatinib arm. Adverse events in the tucatinib arm were primarily low grade and occurred at rates similar to what was observed in the placebo arm. In this report, we characterize the in vitro and in vivo activity of tucatinib alone and in combination with T-DM1 in HER2-amplified preclinical models. In vitro assays demonstrate that tucatinib potently suppresses HER2-meditated signaling pathways, including phosphorylation of HER2, HER3, AKT and ERK. Ex vivo analysis of tucatinib-treated xenografts shows similar suppression of signal transduction pathways. Tucatinib inhibits the proliferation of HER2+ breast cancer (BC) cell lines in vitro with single digit nanomolar potencies, but was inactive in blocking cell proliferation of BC cell lines lacking amplified HER2. In HER2-amplified BC cell line derived- and patient-derived xenograft models, tucatinib suppresses growth of tumors as a single agent and shows improved activity when combined with T-DM1. To evaluate activity and drug penetration in HER2+ metastatic CNS tumors, we developed and characterized a red-shifted luciferase-expressing BT-474 BC cell line which was stereotaxically implanted into the brain. In vivo bioluminescence imaging was used to evaluate the efficacy of tucatinib and T-DM1 in CNS tumors as single agents and in combination. Histological assessment of treated tumors suggests that tucatinib can penetrate the blood-brain tumor barrier to suppress HER2 signaling. These data are further supported by a drug penetration study of CNS-implanted BT-474 tumors. Quantitative autoradiography analysis demonstrates that 14C-labeled tucatinib penetrates the CNS tumor mass with drug concentration levels exceeding measurements in normal brain regions. These data demonstrate that tucatinib is a uniquely selective and highly potent inhibitor of HER2 signaling, with activity against HER2+ breast cancer xenograft and brain metastasis models. Tucatinib is effective against implanted CNS tumors, suggesting that therapeutically relevant drug concentrations are achievable. These preclinical findings are consistent with clinical data showing tucatinib activity in patients with HER2+ MBC with brain metastases and support the continued development of tucatinib in patients with HER2+ BC. Citation Format: Devra J. Olson, Anita Kulukian, Janelle D. Taylor, Margo C. Zaval, Albina Nesterova, Kelly M. Hensley, Michelle L. Ulrich, Nicole S. Stevens, Scott R. Peterson. Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1962.

Abstract 1441: Aspirin in combination with Letrozole or Olaparib induced apoptosis in estrogen receptor-positive T-47D & triple negative MDA-MB-231 breast cancer cell lines

Abstract Breast cancer is the most commonly diagnosed type of cancer and leading cause of cancer-related death among women worldwide. Although there are several breakthroughs in the treatment of breast cancer on the past few decades, high incidence of relapse and progression after conventional therapies is deeply concerning, indicating a great need for developing new therapeutics for breast cancer. Strategies including utilization of combined treatments in order to enhance the efficacy of cancer treatment have been widely developed in recent years, targeting different signaling pathways to inhibit proliferation or induce apoptosis. In the present study, the anti-cancer effects of combinational therapy of Letrozole (Aromatase inhibitor) or Olaparib (PARP inhibitor) with Aspirin, non-steroidal anti- inflammatory drugs (NSAIDs), were examined on well differentiated ER-positive and poorly differentiated triple negative breast cancer cell lines, T-47D and MDA-MB-231, respectively. In addition, The effect of combined treatment was also examined by detecting cell survival, cell cycle analysis, and alterations in signaling pathway that play a role in breast cancer progression such as β -catenin, STAT3, cyclin D1, BAX, c-PARP, wild and Mutant p53. In addition the effect of combined treatments were examined on gene expression levels of Cytochromes; CYP1A1, CYP1B1, Aurora kinases; Aurora-A and Aurora-B were determined by RT-PCR. Our data showed that combined treatments of 50 nm Letrozole or 250 nm Olaparib with 5mm, 2.5mm Aspirin for 48 & 24 h respectively promoted apoptosis induction through enhanced histone release and caspase-3 activation compared to control and cells treated with each drug alone. Furthermore, apoptotic induction was associated with upregulation of Bax and downregulation of cyclin D1, β-catenin, and STAT3 signaling pathways in both tested cell lines compared to control, suggesting that Aspirin may enhance the sensitivity of tested cells to Letrozole or Olaparib in both (ER) positive as well as in triple negative breast cancer cell lines. In addition, cell cycle analysis showed a cell arrest at G0/G1 phase in both cell lines. Interestingly, in T-47D cells, combined treatment of Letrozole with Aspirin significantly upregulated wild p53 at protein level and downregulated CYP1A1, CYP1B1, Aurora-A, and Aurora-B expression at gene level compared to control, suggesting that combined treatment may inhibit breast cancer cell proliferation via down-regulation of Aurora-A and Aurora-B. In MDA- MB-231 cells, combined treatment of Olaparib with Aspirin showed a significant downregulation of Mutant p53 at protein level and CYP1A1, CYP1B1, Aurora-B and upregulation of Aurora-A at gene level compared to control, suggesting that there is a crosstalk between Olaparib/ Aspirin and P53. In conclusion, combined treatment of Letrozole or, Olaparib with Aspirin might be a promising approach to inhibit both ER (+) and triple negative breast cancer cell lines survival and proliferation. Citation Format: Marwa El Sayed El Kamel, Salah Ahmed Sheweita, Ahmed Samir Sultan. Aspirin in combination with Letrozole or Olaparib induced apoptosis in estrogen receptor-positive T-47D & triple negative MDA-MB-231 breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1441.

Unveiling the anticancer and antimycobacterial potentials of bioengineered gold nanoparticles

Synthesis of gold nanoparticles was carried out using Pongammia pinnata (pongam) leaf extract and their anticancer and antimycobacterial activities were studied. Gold nanoparticle formation was confirmed by UV–vis, XRD and HR-TEM. The anticancer efficacies of the biogenic gold nanoparticles were analyzed using cytotoxicity, cell morphology analysis, oxidative DNA damage, apoptosis detection and toxicity studies. Biogenic gold nanoparticles inhibited breast cancer cell line (MCF-7) proliferation with an efficacy of IC50 of 1.85 μg/mL. The antimycobacterial potential of the biogenic gold nanoparticles was screened against M. tuberculosis by Luciferase Reporter Phage (LRP) assay. The gold nanoparticles showed inhibition against sensitive M. tuberculosis with the minimum inhibitory concentration (MIC) of 10 μg/mL whereas no inhibition was found against the rifampicin resistant M. tuberculosis.

Effect of safflower polysaccharide combine cyclopamine on the proliferation and apoptosis of breast cancer cells

Objective To investigate the effect of safflower polysaccharide combine cyclopamine on the proliferation and apoptosis of breast cancer cells. Methods Human breast cancer cell line MDA-MB-231 were cultured, cell proliferation and half maximal inhibitory concentration (IC50) calculation were detected after 0.05, 0.10, 0.20, 0.40, 0.80 mg/ml safflower polysaccharide and 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L cyclopamine were added separately to cells for 24, 48, 72 h by cell counting kit-8 (CCK-8) test; the best concentration of safflower polysaccharide and cyclopamine were choiced according to the IC50; and the next test will be divided into control group, safflower polysaccharide group, cyclopamine group, safflower polysaccharide + cyclopamine group, cells proliferation in four groups were detected by CCK-8 test, apoptosis were detected by flow cytometry, and the expression of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax), Fas, Gli protein was detected by Western blotting. Results IC50 calculation after safflower polysaccharide treated cells for 24, 48, 72 h cell were (0.89±0.04), (0.35±0.03), (0.14±0.02) mg/ml, IC50 calculation after safflower cyclopamine treated cells for 24, 48, 72 h cell were (56.30±1.23), (28.85±1.02), (15.26±0.87) μmol/L, survival rate after safflower polysaccharide and cyclopamine treated cells for 24, 48, 72 h were significantly lower than 0 hour According to the above concentration treatment, 0.8 mg/mL safflower polysaccharide and 20 μmol/L cyclopamine were used for a follow-up study; the cell survival rate in safflower polysaccharide group, cyclopamine group and safflower polysaccharide + cyclopamine group was 58.76%, 56.67%, 24.29%, the control group significantly higher than polysaccharide group (P=0.003), cyclopamine group (P=0.003) and safflower polysaccharide + cyclopamine group (P=0.000), the apoptosis rate in safflower polysaccharide group, cyclopamine group and safflower polysaccharide + cyclopamine group was 19.26%, 16.24%, 26.76%, the control group significantlyhigher than polysaccharide group (P=0.001), cyclopamine group (P=0.001) and safflower polysaccharide + cyclopamine group (P=0.000). cell survival rate in safflower polysaccharide+ cyclopamine group was significantly lower than safflower polysaccharide group (P=0.005)and cyclopamine group (P=0.005), the apoptosis rate in safflower polysaccharide+ cyclopamine group was significantly higher than safflower polysaccharide group (P=0.005) and cyclopamine group (P=0.004); the expression bcl-2 and Gli1 protein in safflower polysaccharide group, cyclopamine group, safflower polysaccharide+ cyclopamine group was significantly lower than the control group, the expression of bax and Fas protein was significantly higher than the control group; the expression of bcl-2, Gli1 protein in safflower polysaccharide+ cyclopamine group was significantly lower than the safflower polysaccharide group and the cyclopamine group, and bax and Fas protein expression was significantly higher than the safflower polysaccharide group and the cyclopamine group. Conclusion Safflower polysaccharide and Hedgehog pathway inhibitors were able to inhibit the proliferation of human breast cancer cell line MDA-MB-231, safflower polysaccharide and cyclopamine combination on cell proliferation and apoptosis were stronger than separate safflower polysaccharide and cyclopamine. Key words: Safflower polysaccharides; Hedgehog signal pathway; Cyclopamine; Breast cancer; Proliferation; Apoptosis

GHRH-R Expression Level under Hyperglycaemic Like Culture Conditions in Breast Cancer Cell Lines

Background: Diabetes mellitus (DM) is a complex disease and increments in its prevalence have been reported in the last few years. DM does not only affect the cells by disturbing glucose metabolism, but also it leads to serious health disorders such as cardiac ischemic diseases, hypertension, and infertility. Intriguingly, DM was also found to mediate cancer development and progression. The exact mechanism of how hyperglycemia affects cancer cells is still obscure. Growth hormone releasing hormone receptor (GHRH-R) is a G-protein coupled receptor that was shown recently to enhance breast cancer cells proliferation. Moreover, a recent work revealed that GHRH-R is upregulated in diabetes mellitus in a rat model. It was of interest to investigate whether hyperglycemic conditions have any impact on the GHRH-R expression in breast cancer cell lines, which might then affect the cell proliferation.Methods: MDA MB 231 and T47D breast cancer cell lines were treated with either 15 mM glucose or 15 mM fructose. The expression of GHRH-R was assessed by western blotting and immunofluorescence. The proliferation of the cells was studied by MTT assays.Results and discussion: The generated data showed that the GHRH-R affected MDA MB 231 and T47D breast cancer cell lines proliferation upon growth hormones treatment but not under hyperglycemic conditions. The results suggested that GHRH-R might mediate breast cancer cells proliferation and survival among other factors.

Abstract 3787: Thyroid hormone induced proliferation of breast cancer cell lines: A novel approach to hormone therapy

Abstract Introduction: The thyroid hormone (TH) pathway influences cell growth and it may be a novel target for breast cancer (BC) therapy. However, various TH receptor (THR) isoforms exist and some have opposing functions thus complicating the role of THs in BC. Our previous work suggests that THR alpha1 (THRα1) promotes TH-mediated BC proliferation. Here we offer a mechanism that explains these findings. We first evaluated the role of THs on BC cell line proliferation using escalating doses of tri-iodothyronine (T3) and thyroxine (T4). Next, propylthiouracil (PTU) was used to block T4 conversion to T3 demonstrating that observed proliferation was TH dependent. Finally, the anti-proliferative efficacy of a THRα1 inhibitor, dronedarone, was used to demonstrate the necessity of THRα1 downstream of TH signalling. Dronedarone was selected because it is an FDA approved anti-arrhythmic drug, which may also serve as a novel anti-cancer agent. Methods: The effect of increasing concentrations of T3 and T4 on the proliferation of 3 BC cell lines (MCF 7, MD-MB-231, BT-474) in 10% charcoal-stripped phenol-free serum were evaluated at 24 and 48 hours following standard MTT-assay protocols. Increasing doses of PTU and dronedarone were added to cells with 200uM T3 or T4 to measure proliferation of MCF-7 and MD-MB-231 cells using MTT assays. Results: There was a statistically significant increase in the proliferation of MCF7, MD-MB-231 and BT-474 cells with the addition of T3 and T4 at 24 and 48 hours in a dose dependent manner. Of note, BT-474 cells had a significantly slower proliferation rate and required 9 days of incubation prior to detection of proliferation. The addition of PTU reduced proliferation by 50% in MCF7 and MD-MB-231 cells in the presence of T4 at roughly 5 mM compared to the presence of T3 which had an IC50 of 12mM. This shows that TH signalling is mediated by T4 conversion to T3 in these cell lines. PTU alone reduced cell proliferation by 50% at concentrations above 12 mM, suggesting that PTU may also act through other mechanisms. Cell proliferation was not affected by the presence of dronedarone alone, but the combination of 10μM dronedarone and T3 or T4 significantly reduced proliferation of both MCF7 and MD-MB-231 cells. These results suggest that THRα1 is required for TH mediated cell proliferation. A library of compounds similar in structure to dronedarone is being designed to offer additional candidates for more potent THRα1 inhibition. The properties and structures of these novel compounds will be presented. Conclusion: The proliferation of several BC cell lines increases in response to exogenous T3 and T4 when grown in hormone depleted growth media. These results demonstrate the role of specific events contributing to TH-mediated cell proliferation, including T4 conversion to T3 and the necessity of downstream THRα1. Ultimately, in conjunction with our previous work, we propose that THRα1 inhibition is a novel target for cancer therapy. Citation Format: Katarzyna Joanna Jerzak, Jessica G. Cockburn, John Hassel, Kathleen I. Pritchard, Sukhbinder K. Dhesy-Thind, Bane Anita. Thyroid hormone induced proliferation of breast cancer cell lines: A novel approach to hormone therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3787.