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Background Breast cancer is the most common cause of the majority of cancer-related deaths in women, among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has been extensively studied as a potent anticancer molecule against various types of cancers. Honeybee products such as the royal jelly (RJ), the nutritive secretion fed to honeybee queens, exhibit a variety of biological activities besides its anticancer effect. However, the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown. Aim To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ, RJ, and their combinations in the MDA-MB-231 cell line. Methods Cells were treated with TQ, RJ, and their combinations for 24 h. Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we determined the half-maximal inhibitory concentration of TQ. Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions. Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ, RJ, and their combination. Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation. Results TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration. RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5 µg/mL. High doses of RJ (200 µg/mL) had greater toxicity against MDA-MB-231 cells. Interestingly, the inhibition of cell viability was most pronounced in response to 15 µmol/L TQ and 5 µg/mL RJ. A dose of 15 µmol/L TQ caused a significant increase in the PreG1 population, while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations. TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ. In contrast, no significant regulation of Ki67 expression was observed, indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation. Conclusion This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells, which could confer an advantage for cancer therapy.

DNAJC25 gene is a member of the HSP40 (DNAJ) family, and it was suggested as a tumor suppressor gene in hepatocellular carcinoma. The aim of this study was to analyze the expression, genetic/epigenetic alterations, and prognostic value of the DNAJC25 gene in breast cancer. DNAJC25 transcript levels are upregulated in BT-20 and ZR-75-1 cell lines and downregulated in MDA-MB-231 cell line compared to the non-tumorigenic mammary epithelial cell line (MCF 10A) (P< 0.05). According to UALCAN in-silico tool, clinical breast cancer samples show significantly reduced levels of DNAJC25 mRNA levels relative to the normal samples (P=1.47e-02). The Kaplan–Meier plotter tool shows that high DNAJC25 expression is favorable for post-progression survival in breast cancer (P=0.0035). Point mutations or copy number variations of DNAJC25 are uncommon in clinical breast cancer samples. Combined bisulfite restriction analysis (COBRA) results showed that DNAJC25 promoter is not methylated in breast cell lines. Promoter hypomethylation was also observed in normal and tumor clinical samples (Beta-value < 0.25). In conclusion, DNAJC25 is suggested as a tumor suppressor candidate having limited biomarker potential in breast cancer. Functional studies are essential to reveal its role in breast carcinogenesis.

Background Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia. Methods RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo. Results SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3. Conclusions Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.

To construct a CXCR4 specific recombinant plasmid vector and study its inhibiting effect on invasion capacity in vitro of human breast cancer MDA-MB-231 cell line and its metastatic potential to the lung in nude mice.A CXCR4 specific recombinant plasmid vector was constructed and transfected into the cultured MDA-MB-231 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4, respectively. Invasion capability in vitro of the cells was evaluated by Boyden chamber. The cell proliferation capacity was detected by MTT method. The nude mouse model of lung metastasis was established by injection of MDA-MB-231 cells into the tail vein. The animals were sacrificed at 6 weeks after the tumor cells injection. Whole lung tissues were harvested, embedded in paraffin, sectioned serially, and the HE-stained paraffin sections were examined pathologically to evaluate the presence and number of metastatic tumors.The CXCR4 mRNA expression rate was 29.5% +/- 3.8% in the CXCR4-shRNA group, significantly lower than that of the control group (69.7% +/- 2.6%, P0.01) and mock-control group (67.8% +/- 3.5%, P0.01). The CXCR4 protein expression rate was 15.4% +/- 1.1% in the CXCR4-shRNA group, significantly lower than that of the control group (39.0% +/- 2.4%, P0.01) and mock-control group (35.9% +/- 3.9%, P0.01). Silencing of CXCR4 by shRNA lead to a significant decrease in breast cancer cell invasion and proliferation capacity in vitro. Furthermore, tumor cells with CXCR4 shRNA permanent transfcetion had a much lower lung metastatic potential in nude mice than control cells and mock control cells in vivo.CXCR4 shRNA can inhibit the expression of CXCR4 and decrease the invasion and lung metastatic potential of human breast cancer cells.

Aistė Savukaitytė,1 Greta Gudoitytė,1 Agnė Bartnykaitė,1 Rasa Ugenskienė,1,2 Elona Juozaitytė3 1Oncology Research Laboratory, Institute of Oncology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Biology Systems and Genetic Research, Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Department of Oncology and Hematology, Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas, LithuaniaCorrespondence: Aistė Savukaitytė Email aiste.savukaityte@lsmuni.ltBackground: Mutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action.Methods: MDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin.Results: We show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells.Conclusion: The current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.Keywords: aspirin, breast cancer, REDD1, mTORC1 signaling, 4E-BP1

Aistė Savukaitytė,1 Greta Gudoitytė,1 Agnė Bartnykaitė,1 Rasa Ugenskienė,1,2 Elona Juozaitytė3 1Oncology Research Laboratory, Institute of Oncology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Biology Systems and Genetic Research, Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Department of Oncology and Hematology, Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas, LithuaniaCorrespondence: Aistė Savukaitytė Email aiste.savukaityte@lsmuni.ltBackground: Mutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action.Methods: MDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin.Results: We show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells.Conclusion: The current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.Keywords: aspirin, breast cancer, REDD1, mTORC1 signaling, 4E-BP1

Objectives: Triple negative breast cancer (TNBC) is defined by lack of expression in estrogen (ER) and progesterone receptors (PR) as well as human epidermal growth factor receptor 2 (HER2), which accounts for 10-15% of all breast cancers. TNBC is a more aggressive histological subtype with worse prognosis when compared to other subtypes due to limited therapeutic targets for hormonal and HER2 receptors. With limited treatment options and ineffective targeted therapy, there is an urgent need for the development of novel therapeutic targets to treat this malignant form of breast cancer. Methods: Focused on the previously identified TNBC biomarker miR-199a-5p by our group, cells were transfected with 199a-5p mimic for functional studies. MTT and wound healing assay were performed to investigate the cell proliferation and migration ability. Immunofluorescence staining was introduced to study TWIST1 and E-cadherin expression in miR199a-5p stable transfected cells. Aldehyde dehydrogenase (ALDH) expression level was compared between breast cancers and normal control cases’ plasma by using real-time RT-PCR. We further used ALDH activity assay to evaluate the stem cell population. In vivo study was also carried out in nude mice to study tumorigenicity. Results: Cell proliferation assay showed that overexpression of miR-199a-5p significantly inhibited cell growth (p = 0.0008). Wound healing assay indicated the ectopic expression of miR-199a-5p can inhibit cell migration compared to control group. In miR-199a-5p stable transfected MDA-MB-231 cell line, increased translocation of Twist 1 protein from nucleus to cytoplasm and increased expression of E-cadherin was detected by immunofluorescence staining. ALDH activity assay result indicated that the proportion of ALDH(BR+) cells decreased significantly from 17.21% to 8.95% in the miR-199a-5p transfected cells when compared to the control group (p = 0.0279). Consistent with in vitro study, the well-recognized breast cancer stem-cell marker, ALDH1A, was significantly upregulated in the plasma of breast cancer patients than in healthy controls. Moreover, in vivo study also revealed that stable MDA-MB-231 cell line with ectopic miR199a-5p expression resulted in smaller volume of tumors in nude mice. Conclusions: Our data implicate that miR-199a-5p plays a tumor suppressive role in the pathogenesis of TNBC, which may due to the inhibition of stemness characteristics in breast cancer cells. These findings suggest that miR-199a-5p involved in TNBC stemness-related features and thus provide insights in development of the novel therapeutic strategies for this highly malignant breast cancer. Citation Format: Jiawei Chen, Man-Ting Siu, John Chi-Wang Ho, Vivian Yvonne Shin, Ava Kwong. MiR-199a-5p confers tumor suppressive role by inhibiting stenmness characteristics in triple-negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2015-3105

Ligustrazine (TMP) is a natural pyrazine alkaloid extracted from the roots of Ligusticum Chuanxiong Hort, which has the potential as an antitumor agent. A series of 33 ligustrazine–heterocycle (TMPH) derivatives were designed, synthesized, and investigated via antitumor screening assays, molecular docking analysis, and prediction of drug-like properties. TMP was attached to other heterocyclic derivatives by an 8–12 methylene alkyl chain as a linker to obtain 33 TMPH derivatives. The structures were confirmed by 1H-NMR, 13C-NMR, and high-resolution mass spectroscopy spectral (HR-MS) data. The antiproliferative activity against human breast cancer MCF-7, MDA-MB-231, mouse breast cancer 4T1, mouse fibroblast L929, and human umbilical vein endothelial HUVEC cell lines was evaluated by MTT assay. Compound 12–9 displayed significant inhibitory activity with IC50 values in the low micromolar range (0.84 ± 0.02 µM against the MDA-MB-231 cell line). The antitumor effects of compound 12–9 were further evaluated by plate cloning, Hoechst 33 342 staining, and annexin V-FITC/PI staining. The results indicated that compound 12–9 inhibited the proliferation and apoptosis of breast cancer cells. Furthermore, molecular docking of compound 12–9 into the active site of the Bcl-2, CASP-3, and PSMB5 target proteins was performed to explore the probable binding mode. The 33 newly synthesized compounds were predicted to have good drug-like properties in a theoretical study. Overall, these results indicated that compound 12–9 inhibited cell proliferation through PSMB5 and apoptosis through Bcl-2/CASP-3 apoptotic signaling pathways and had good drug-like properties. These results provided more information, and key precursor lead derivatives, in the search for effective bioactive components from Chinese natural medicines.

Breast cancer is considered the type of cancer that most affects women in the world. The triple negative breast cancer is considered aggressive with poor prognosis. In the 1930s Russian researchers observed that T. cruzi has tropism for tumor cells. Since then, this research field has been subject of a numerous of researches. Here, we proposed to investigate the impact of T. cruzi infection on proliferation and migration of triple negative breast cancer cell line (MDA-MB-231). T. cruzi showed high invasion and multiplication rate in MDA-MB-231 cell line. The infection promoted the multiplication of MDA-MB-231 cell, continuous cell lysis throughout of days of in vitro infection and impaired MDA-MB-231 cell migration. Taken together, these results demonstrated the high susceptibility of MDA-MB-231 cell to T. cruzi and suggested that molecules from T. cruzi may impair host cell migration with potential use to avoid metastasis.

Hypoxia plays a vital role in tumor microenvironment by allowing development and maintenance of cancer cells thereby led to major hindrance for effective anticancer therapy and main reason for failure of most anticancer drugs. We herein investigated the therapeutic efficacy and molecular mechanism of action of aqua-(2-formylbenzoato) triphenyltin (IV) compound (OTC) in MDA-MB-231 cell line. Cobalt chloride induced hypoxic MDA-MB-231 cells treated with OTC were used to access cytotoxicity, ROS, cellular apoptosis, and cell cycle progression. Further, expression of HIF-1α and VEGF, as well as apoptotic proteins like p53, Bax, Bcl-2 and caspase 3 were assessed. The findings indicated that OTC is more effective towards CoCl

Chemotherapeutics such as platinum-based drugs are commonly used to treat several cancer types, but unfortunately, their use is limited by several side effects, such as high degradation of the drug before entering the cells, off-target organ toxicity and development of drug resistance. An interesting strategy to overcome such limitations is the development of nanocarriers that could enhance cellular accumulation in target cells in addition to decreasing associated drug toxicity in normal cells. Here, we aim to prepare and characterize a graphene-oxide-based 2D nanoplatform functionalised using highly branched, eight-arm polyethylene-glycol, which, owing to its high number of available functional groups, offers considerable loading capacity over its linear modalities and represents a highly potent nanodelivery platform as a versatile system in cancer therapy. The obtained results show that theGO@PEGcarrier allows for the use of lower amounts of Pt drug compared to a Pt-free complex while achieving similar effects. The nanoplatform accomplishes very good cellular proliferation inhibition in osteosarcoma, which is strictly related to increased cellular uptake. This enhanced cellular internalization is also observed in glioblastoma, although it is less pronounced due to differences in metabolism compared to osteosarcoma. The proposedGO@PEGnanoplatform is also promising for the inhibition of migration, especially in highly invasive breast carcinoma (i.e., MDA-MB-231 cell line), neutralizing the metastatic process. TheGO@PEGnanoplatform thus represents an interesting tool in cancer treatment that can be specifically tailored to target different cancers.

Despite the fact of availability of several treatments for breast cancer, most of them fail to attain the desired therapeutic response due to their poor bioavailability, high doses, non-selectivity and as a result systemic toxicity. Here in an attempt made to study the transdermal effect of leflunomide (LEF) against breast cancer. In order to improve the poor physicochemical properties of LEF, it was loaded into cubosomes. Cubosomes were prepared by the emulsification method. Colloidal characteristics of cubosomes including particle size, ζ-potential, entrapment efficiency, in-vitro release profile and ex-vivo permeation were studied. In addition, morphology, stability, cytotoxicity and cell uptake in MDA-MB-231 cell line were carried out for the selected cubosomal formulation. The selected LEF loaded cubosomal formulation showed a small particle size (168 ± 1.08) with narrow size distribution (PI 0.186 ± 0.125) and negative ζ potential (-25.5 ± 0.98). Its Entrapment efficiency (EE%) was 93.2% and showed sustained release profile that extended for 24 h. The selected formulation showed stability when stored at 25 °C for three months in terms of size and EE%. TEM images illustrated the cubic structure of the cubosome. Cell culture results revealed the superiority of LEF cubosomes compared to LEF suspension in their cytotoxic effects with an IC50 close to that of doxorubicin. Furthermore, LEF cell uptake was significantly higher for LEF cubosomes. This may be attributed to the effect of nano-encapsulation on enhancing drug pharmacological effects and uptake indicating the potential usefulness of LEF cubosomes for breast cancer management.

The chemical contents, antibiofilm and anticancer effects of different propolis extracts (petroleum ether, chloroform, ethyl acetate and methanol) collected from Mila (Algeria) were investigated in this study. Chemical analysis showed the presence flavonoids as a major compound. The highest antibiofilm activity was observed at the 200 µg/ml methanol fraction (92%). Florescent microscope images also confirmed antibiofilm effect. Our results showed that the structure of biofilm treated with propolis was degraded and there were no water channels seen in the biofilm structure. In addition, the density of dead cells in the biofilm was also noted with fluorescent staining. According to FTIR analysis, it was found that propolis caused some changes in macromolecules on the cell surface. Cytotoxic activity of propolis (EC50: 97–117 µg/ml) was determined by MTT test. Propolis extracts caused a significant increase in mRNA levels of proteins involved in tumor suppression and apoptosis in the MDA-MB-231 cell line and induced apoptosis. Western blot analysis confirmed these results. As a result, it can be said that Algerian propolis has anticancer and antibiofilm effective compounds.

Both bisphosphonates and radiotherapy are highly effective for the management of bone metastases. Our in vitro study examined the cytotoxic effects resulting from combinations of ibandronate and ionising radiations (RX) in various sequences on breast cancer cells. Single radiation doses were given before, at halftime of, or after acute ibandronate incubation (48 h). Single or fractionated radiation doses were applied at the end of chronic ibandronate incubation (5 weeks). Combination of acute ibandronate exposure and single radiation doses led to synergistic cytotoxic effects in MDA-MB-231 cell line, but only with low ibandronate concentrations in MCF-7 cell line. In both cell lines, synergy was more marked when ibandronate followed RX. After long-term ibandronate exposure, only high single radiation doses induced synergistic effects in MDA-MB-231 cell line. Synergy was only detected with low ibandronate concentrations in MCF-7 cell line. In both cell lines, fractionated radiation doses exerted similar effects. The combination of ibandronate with radiation can exert synergistic effects on the inhibition of breast cancer cells growth, depending on cell line, drug sequence and dosage. Our data might provide a rationale for associating bisphosphonates and radiotherapy for the treatment of bone metastases from breast cancer.

1- and 1,5-Aminoalkylamine substituted anthraquinones (AAQs, 1C3 and 1,5C3) were peptide coupled to 1-, 2-, and 3-pyrrole lexitropsins to generate compounds that incorporated both DNA minor groove and intercalating moieties. The corresponding platinum(II) amidine complexes were synthesized through a synthetically facile amine-to-platinum mediated nitrile 'Click' reaction. The precursors as well as the corresponding platinum(II) complexes were biologically evaluated in 2D monolayer cells and 3D tumour cell models. Despite having cellular accumulation levels that were up to five-fold lower than that of cisplatin, the platinum complexes had cytotoxicities that were only three-fold lower. Accumulation was lowest for the complexes with two or three pyrrole groups, but the latter was the most active of the complexes exceeding the activity of cisplatin in the MDA-MB-231 cell line. All compounds showed moderate to good penetration into spheroids of DLD-1 cells with the distributions being consistent with active uptake of the pyrrole containing complexes in regions of the spheroids starved of nutrients.

Background To synthesize acetylated low anticoagulant low molecular weight heparin (ALMWH) and to detect its antineoplastic activity. Methods We obtained Low anticoagulant low molecular weight heparin (LMWH) by splitting unfractionated heparin (UFH) with sodium periodate oxidation and sodium borohydride reduction, then the LMWH was subjected to acetylate catalyzed by dicyclohexylcarbodiimide and dimethylaminopyridine to produce ALMWH. The anti-proliferative activities were determined on MDA-MB-231 human breast cancer cells in vitro. Results ALMWH exhibited stranger anti-proliferative activity Compared with LMWH, In the MDA-MB-231 cell line, the growth of MDA-MB-231 cells with IC50 of 22.16 µM at 48 h in a concentration-dependent and time-dependent manner, ALMWH produced stronger inhibitory effects especially when it was used in low concentrations. By the use of bulky catalysts, the acetylation site in the molecular chain of low molecular weight heparin with a high selectivity, the synthesis process of Low anticoagulant low molecular weight heparin can be easily controlled. Therefore, large scale industrial production can be carried out. Conclusions The synthesized ALMWH possesses a high anti-proliferative activity, Chemical modification of structure can endow LMWH with a high antiproliferative activities. ALMWH is expected to enter clinical trials due to its high druggability. Simultaneously, this study provides a basic method for screening of antineoplastic drug with low toxicity.

5-Nitroisatin-4-(1-(2-pyridyl)piperazinyl)-3-thiosemicarbazone (Nitistpyrdlpz) and its Cu(II) complex were synthesized and characterized by CHN and thermal analysis and spectroscopic measurements viz. UV-vis, FTIR, 1H NMR, 13C NMR, ESI-HRMS, PXRD and EPR. In the complex, copper(II) ion is coordinated by terdentate thiosemicarbazone anion and one chloride ion in a distorted square planar geometry. The synthesized compounds against breast cancer cell lines; MCF-7 and MDA-MB-231 and epidermoid carcinoma; A431 showed that the complex contributed to reduce the percentage of cell viability toward all the tested cell lines but remarkable contribution toward MDA-MB-231 cell line. The IC50 of the complex and free ligand was found in the range of IC50 0.85-1.24 μM and IC50 3.28-3.53 μM, respectively. Among those cell lines, the complex may be the better anticancer agent toward MDA-MB-231 because of its action at micromolar concentration (IC50 0.85 μM).

The aim of the research. To examine the proteomic profi le of breast cancer exosomes. Material and methods. Cell lines used for this study were MDA-MB-231 female epithelial breast cancer cells (ATCC HTB-26) and MCF10A non-tumorigenic epithelial breast tissue cells. MVs were isolated using diff erential ultracentrifugation. Samples were lysed, reduced, alkylated, digested, and analyzed by an Orbitrap Fusion mass spectrometer. MS raw fi les were analyzed using MaxQuant version 1.6.12.0. Peptides were searched against the human UniProt FASTA database using the Andromeda search engine, integrated into MaxQuant. Results. MVs derived from MCF10A and MDA-MB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. MVs derived from MCF10A and MDAMB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. Th e unique MDA-MB-231 MV proteins were searched against the DisGeNET human diseases database. Out of 972 MDA-MB-231 MV proteins, 112 were cancer-related while 32 were specifi cally associated with BC. In the MDA-MB-231 MV proteome, 23 Wnt signaling pathway proteins were identifi ed based on their GO biological process. Proteomic analysis identifi ed enzymes OAT, TALDO1, and BLMH were only in MVs from metastatic MDA-MB-231 cell line. The specific activity of OAT and TALDO1 was higher in MV fractions of MDA-MB-231 in comparison to the non-cancerous MCF10A cell line-derived MVs. Th ese fi ndings might suggest that these enzymes might play a role in BC. In our present study, we found that some enzymes identifi ed from MV fractions were already proposed to play a role in cancer therapy as therapeutic targets (OAT, TALDO1) and resistance against chemotherapy agents (BLMH).

One of the best strategies to circumvent drug resistance is the employment of nanocarriers. For the current study, we have employed a nanoemulsion formulation of paclitaxel (PTX) to bypass drug resistance in the MDA-MB-231 cell line and impedance sensing biosensors to determine the exact time that PTX-NE induced apoptosis. Our MTT results demonstrated that PTX treatment could not reduce MDA-MB-231 cell viability to IC50 even after three days. However, the employment of the reagent TPGS (inhibitor of drug resistance) combined with paclitaxel could partially obviate PTX resistance. Next, the nanoemulsion form of PTX (PTX-NE) was fabricated employing the essential oil of the Satureja khuzestanica plant and was characterized using DLS and TEM methods. Our data showed that after 72 hours, PTX-NE at 250 nM concentration could induce a 50% reduction in cell viability. Moreover, annexin/PI and cell cycle analysis confirmed the apoptotic effect of PTX-NE on cancer cells. Lastly, we measured the impedance of MDA-MB-231 cells treated with the free and nanoemulsion forms of PTX. A significant decrease in the mean impedance of PTX-NE treated cells could be observed after 40 hours. To conclude, we have demonstrated here that PTX-NE could circumvent resistance and induce apoptosis in PTX-resistant breast cancer cells, which could be inferred from their impedance measurement.

This work describes the synthesis of three new ruthenium(ii) complexes with gallic acid and derivatives of the general formula [Ru(L)(dppb)(bipy)]PF6, where L = gallate (GAC), benzoate (BAC), and esterified-gallate (EGA), bipy = 2,2'-bipyridine and dppb = 1,4-bis(diphenylphosphino)butane. The complexes were characterized by elemental analysis, molar conductivity, NMR, cyclic voltammetry, UV-vis and IR spectroscopy, and two of them by X-ray crystallography. Cell viability assays show promising results, indicating higher cytotoxicity of the complexes in MDA-MB-231 cells, a triple-negative breast cancer (TNBC) cell line, compared with the hormone-dependent MCF-7 cell line. Studies in vitro with the MDA-MB-231 cell line showed that only Ru(BAC) and Ru(GAC) interacted with BSA. Besides that, the Ru(GAC) complex, which has a polyphenolic acid, interacted in an apo-Tf structure and function dependent manner and it was able to inhibit the formation of reactive oxygen species. Ru(GAC) was able to cause damage to the cellular cytoskeleton leading to inhibition of some cellular processes of TNBC cells, such as invasion, migration, and adhesion.

Background: In this study, we aimed to determine the gene expressions of prostaglandin endoperoxide H synthase 2 (PTGS2), calreticulin (CALR), and keratin-19 (KRT19) genes at transcriptional level in MDA-MB-231 breast cancer cell line.Materials and Methods: RNA was isolated from MDA-MB-231 breast cancer cells grown in culture medium, followed by cDNA synthesis. Real-time PCR with specific primers for PTGS2, CALR and KRT19 genes was used to determine the expression of these genes at the transcriptional level.Results: In the MDA-MB-231 cell line, a 14.92-fold increase in PTGS2 gene expression, a 1.45-fold increase in CALR gene expression and a 6.72-fold increase in expression of the KRT19 gene were found.Conclusions: It is predicted that the KRT19, CALR, PTGS2 genes, which are known to play a role in biological processes such as apoptosis resistance, metastasis, and angiogenesis in different solid cancers, may also play a role in the development of breast cancer and will have prognostic importance with further studies.Key words: Breast cancer, PTGS2 gene, CALR gene, KRT19 gene, Amaç: Bu çalışmada MDA-MB-231 meme kanser hücre hattında prostaglandin endoperoksid H sentaz 2 (PTGS2), kalretikulin (CALR) ve keratin-19 (KRT19) genlerinin transkripsiyon düzeyindeki gen anlatımlarının belirlenmesi amaçlanmıştır.Materyal ve metod: Kültür ortamında çoğaltılan MDA-MB-231 meme kanser hücrelerinden RNA izolasyonu yapılmış ardından cDNA sentezi gerçekleştirilmiştir. PTGS2, CALR ve KRT19 genlerine spesifik primerler ile eş zamanlı PCR yapılarak, bu genlerin ifadesi transkripsiyonel seviyede belirlenmiştir.Bulgular: MDA-MB-231 hücre hattında PTGS2 gen ifadesinde 14,92 kat; CALR gen ifadesinde 1,45 kat; KRT19 geninin ifadesinde ise 6,72 kat artış olduğu saptanmıştır.Sonuç: Farklı solid kanserlerde, apoptoz direnci, metastaz, anjiyogenez gibi biyolojik süreçlerde rol aldığı bilinen KRT19, CALR, PTGS2 genlerinin meme kanseri gelişiminde de rol alabileceği ve ileride yapılacak detaylı çalışmalarla prognostik öneme sahip olacağı ön görülmektedir.Anahtar Kelimeler: Meme kanseri, PTGS2 geni, CALR geni, KRT19 geni

Mesoporous materials have attracted extensive consideration in the field of drug delivery. The targeted release of anti-neoplastic agents depends on the carrier used to transport such drugs in the body. In this study, mesoporous bioactive glass nanoparticles (MBGN) of an average size of 120 ± 10 nm were used to load and release silibinin, which is a natural flavonolignan used as anticancer agent and tumor suppressor. Mesoporous bioactive glass nanoparticles were characterized in terms of morphology and release potential after being loaded with silibinin. The loading and release amounts of silibinin were quantified using UV-VIS spectrophotometer at λmax = 280 nm in PBS buffer solution (pH 7.4). It was found that MBGN have a maximum loading efficiency of 61% against optimal silibinin concentration (i.e. 40 μg/ml silibinin in PBS buffer solution (pH 7.4)). The drug was released in a sustained manner from loaded mesoporous bioactive glass nanoparticles and a release of 18 μg/ml silibinin was measured during the first 24 h post-drug loading. Further, the chemotherapeutic effect of silibinin was tested on metastatic MDA-MB-231 breast cells and the non-metastatic breast cell line MCF-10A at two different concentrations (9.0 μg/ml and 18 μg/ml). As our results show, silibinin exhibits a chemotherapeutic potential towards MDA-MB-231 cell line, which is a much lower concentration compared to values reported in literature. According to our knowledge, this is the first study elucidating the use of MBGN as a carrier for silibinin, as well as testing its lethal effect in contact with breast neoplastic cells. Taken collectively, the results of the present study suggest that silibinin-loaded MBGN can be applied as an effective drug delivery system to cause a chemo preventive response which has the potential to be beneficial for the treatment of breast cancer.

Background Breast cancer (BC) is the leading cause of tumor-related death in women worldwide, but its pathogenesis is not clear. The efficient screening of new therapeutic targets for BC through bioinformatics and biological experimental techniques has become a hot topic in BC research. Methods The bioinformatics method was used to analyze the gene chips and obtain the hub genes, playing an important role in the development of BC. The biological processes (BP) involved in the hub genes were analyzed by Bingo, and the impact of each hub gene on disease-free survival (DFS) and overall survival (OS) in BC patients was evaluated in the Kaplan-Meier Plotter database. The expression of DNAJB4, the hub gene with the greatest degree and having an effect on the prognosis of BC patients, was detected in BC cell lines and clinicopathological specimens. And DNAJB4 was selected for further biological experiments and clinical prognosis verification. Results Ten hub genes including DNAJB4, the greatest degree genes, were found by bioinformatics analysis of BC gene chips. DNAJB4 expressions in both BC cell lines and clinicopathological specimens were detected and the results showed that DNAJB4 was significantly down-regulated in BC cell lines and tissues. After interfering with the expression of DNAJB4, it was found that the invasion and migration ability of MDA-MB-231 cell line was significantly enhanced in vitro. The clinical survival data of BC patients showed that patients with high DNAJB4 expression had longer DFS. Conclusions DNAJB4 may be a tumor suppressor gene in BC as it could regulate invasion and migration of BC cells and its expression level is related to the prognosis of BC patients. Nevertheless, further researches are still necessary to verify its role in BC so as to provide evidences for clinical guidance regarding diagnosis and treatment.

This study aimed to investigate the effect of Catharanthus roseus L. (C. roseus) leaf extract on the migration and invasion of MDA-MB-231 cell line and elucidate the molecular mechanisms of action. Effect of the extract on cell viability was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazoliumbromide) assay. Anti-migratory and anti-invasive effects were evaluated using scratch and Transwell assays. Effect on the levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9 was determined using ELISA and gelatin zymography. Furthermore, changes in the expression of 84 genes commonly involved in cell motility were assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and cell motility RT2 profiler PCR array. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID. Using STRING and Cytoscape software, hub genes were determined. The extract significantly (p

Four ball-type phthalocyanines (H2Pc, MgPc, CoPc, and ZnPc) containing 4-tert-butylcatechol substituents at alpha-positions were synthesized from 3,3′-(4-tert-butyl-1,2-phenylene)bis(oxy)diphthalonitrile. The structures were elucidated by elemental analysis and ultraviolet-visible, Fourier-transform infrared, proton nuclear magnetic resonance, and Maldi-Tof mass spectroscopies. The photophysicochemical properties of H2Pc, MgPc, and ZnPc were investigated in dimethylsulphoxide. The fluorescence quantum yields (ΦF), triplet state quantum yields (ΦT), and singlet oxygen quantum yields (ΦΔ) with respect to H2Pc, MgPc and ZnPc were experimentally obtained. Furthermore, the degradation quantum yield (Φd) was evaluated for all complexes. Finally, the magnetic circular dichroism spectra of the four Pcs were obtained. Among these ball-type phthalocyanines, MgPc had the highest triplet lifetime and ZnPc had the highest singlet oxygen quantum yield. Moreover, the in vitro antiproliferative effect of ball-type phthalocyanines (H2Pc, MgPc, CoPc, and ZnPc) was evaluated on MCF-7 cell line originated from luminal A breast cancer and MDA-MB-231 cell line originated from triple negative breast cancer. For this purpose, cell viability MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used. For all compounds, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, 102.4 µM concentrations were applied to both cell lines. There was a significant decrease in cell viability for MCF-7 and MDA-MB-231 cells (p

Black chickpea is a good source of bioactive compounds, particularly isoflavones. Sprouting improves nutraceutical value in chickpea seeds. This study aimed to explore the role of sprouting of black chickpea seeds on the synthesis of isoflavones and evaluate the impact of the soluble isoflavone on cellular antioxidant activity (CAA) and antiproliferative activity in breast cancer cells. Isoflavones were identified and quantified by HPLC-UV-MS. The CAA and antiproliferative activity were determined in HepG2 cells and MDA-MB-231 cancer cells, correspondingly. In sprouted black chickpea, six isoflavones (formononetin, biochanin-A, and its glycosides) were identified and the total isoflavones content increased (0.31 to 35.72 µgBA/mg of extract). The CAA was increased five times from 137.2 to 788.2 µMEQ/100 g of sample. The bioactive compounds in sprouted chickpea decreased the proliferation of MDA-MB-231 cell line. Also caused morphological changes such as cell shrinkage, rounding and nuclear fragmentation. The results herein suggest that bioactive compounds, as isoflavones, in sprouted black chickpea showed a potential antioxidant and antiproliferative activity. Therefore, it may be considered as a value-added product or ingredient for produce functional foods.

In this study, antiproliferative effects of the anti-EGFR monoclonal antibody Cetuximab were evaluated using MDA-MB-231 cell line originated from triple negative breast cancer. As cell kinetic parameters, we evaluated Cell index, mitotic index, labeling index and apoptotic index. For this purpose, 20 μM, 45 μM and 60 μM Cetuximab concentrations were applied to the cells using the real-time cell analysis system (xCelligence DP) and IC50 values were determined. IC50 concentrations were used for all other parameters. According to experimental results, Cetuximab administration inhibited cell kinetics of MDA-MB-231 cells. xCelligence DP instrument detected IC50 concentrations of Cetuximab for cell line. These values were 45 μM for MDA-MB-231 cells. When these IC50 value applied to cells, significant decrease was detected in mitotic index, labelling index and significant increase was detected in apoptotic index for experimental groups. Student’s t tests for paired samples were used to assign statistical significance. p

Prodigiosin, a secondary metabolite red pigment produced by Serratia marcescens, has an interesting apoptotic efficacy against cancer cell lines with low or no toxicity on normal cells. HSP90α is known as a crucial and multimodal target in the treatment of TNBC. Our research attempts to assess the therapeutic potential of prodigiosin/PU-H71 combination on MDA-MB-231 cell line. The transcription and protein expression levels of different signalling pathways were assessed. Treatment of TNBC cells with both drugs resulted in a decrease of the number of adherent cells with apoptotic effects. Prodigiosin/PU-H71 combination increased the levels of caspases 3,8 and 9 and decreased the levels of mTOR expression. Additionally, there was a remarkable decrease of HSP90α transcription and expression levels upon treatment with combined therapy. Also, EGFR and VEGF expression levels decreased. This is the first study to show that prodigiosin/PU-H71 combination had potent cytotoxicity on MDA-MB-231 cells; proving to play a paramount role in interfering with key signalling pathways in TNBC. Interestingly, prodigiosin might be a potential anticancer agent to increase the sensitivity of TNBC cells to apoptosis. This study provides a new basis for upcoming studies to overcome drug resistance in TNBC cells.

Anti-angiogenesis is an important and promising strategy in cancer therapy. However, the current methods using anti-vascular endothelial growth factor A (VEGFA) antibodies or inhibitors targeting VEGFA receptors are not as efficient as expected partly due to their low efficiencies in blocking VEGFA signaling in vivo. Until now, there is still no method to effectively block VEGFA production in cancer cells from the very beginning, i.e., from the transcriptional level. Here, we aimed to find bioactive small molecules to block VEGFA transcription. Methods: We screened our natural compound pool containing 330 small molecules derived from Chinese traditional herbs for small molecules activating the expression of seryl-tRNA synthetase (SerRS), which is a newly identified potent transcriptional repressor of VEGFA, by a cell-based screening system in MDA-MB-231 cell line. The activities of the candidate molecules on regulating SerRS and VEGFA expression were first tested in breast cancer cells. We next investigated the antiangiogenic activity in vivo by testing the effects of candidate drugs on the vascular development in zebrafish and by matrigel plug angiogenesis assay in mice. We further examined the antitumor activities of candidate drugs in two triple-negative breast cancer (TNBC)-bearing mouse models. Furthermore, streptavidin-biotin affinity pull-down assay, coimmunoprecipitation assays, docking analysis and chromatin immunoprecipitation were performed to identify the direct targets of candidate drugs. Results: We identified emodin that could greatly increase SerRS expression in TNBC cells, consequently reducing VEGFA transcription. Emodin potently inhibited vascular development of zebrafish and blocked tumor angiogenesis in TNBC-bearing mice, greatly improving the survival. We also identified nuclear receptor corepressor 2 (NCOR2) to be the direct target of emodin. Once bound by emodin, NCOR2 got released from SerRS promoter, resulting in the activation of SerRS expression and eventually the suppression of VEGFA transcription. Conclusion: We discovered a herb-sourced small molecule emodin with the potential for the therapy of TNBC by targeting transcriptional regulators NCOR2 and SerRS to suppress VEGFA transcription and tumor angiogenesis.

The absence of receptors in triple-negative breast cancer limits therapeutic choices utilized in clinical management of the disease. Doxorubicin is an important member of therapeutic regimens that is hindered by emergence of resistance. The current work aim to investigate of therapeutic potential of single and combinations of siRNA molecules designed for silencing STAT 3, Notch-1, and β-catenin genes in wild type and doxorubicin resistant MDA-MB-231 triple negative breast cancer cell line. Doxorubicin resistant MDA-MB-231 cell line was developed and characterized for the expression of multidrug resistance-related genes, CD44/CD24 markers, inflammatory cytokines, and the expression of STAT 3, Notch-1, and β-catenin targeted genes. Further, the effect of single and combinations of siRNA on cell viability and chemosensitivity of both wild type MDA-MB-231 cells (MDA-MB-231/WT) and doxorubicin resistant MDA-MB-231 cells (MDA-MB-231/DR250) were assessed by MTT assay. The IC50 of doxorubicin was 10-folds higher in MDA-MB-231/DR250 resistant cells compared to MDA-MB-231/WT control cells, 1.53 ± 0.24 μM compared to 0.16 ± 0.02 μM, respectively. The expression of targeted genes was higher in resistant cells compared to control cells, 3.6 ± 0.16 folds increase in β-catenin, 2.7 ± 0.09 folds increase in Notch-1, and 1.8 ± 0.09 folds increase in STAT-3. Following treatment with siRNAs, there was a variable reduction in mRNA expression of each of the targeted genes compared to scrambled siRNA and a reduction in IC50 in both cell lines. The effect of a combination of three genes produced the largest reduction in IC50 in resistant cell line. Our study showed that the silencing of single and multiple genes involved in drug resistance and tumor progression by siRNA can enhance the chemosensitivity of cancer cells to conventional chemotherapy.

Cancer has been considered a metabolic disease in recent years. Compared to normal cells, cancer cells rewire metabolic pathways to sustain their outgrowth. While growing fast, cancer cells in the core region of a solid tumor encounter metabolic stress induced by hypoxia and insufficient nutrition, and mitochondrial dynamics has a profound effect on the survival of cancer cells. However, how mitochondrial dynamics affects cancer cell survival under metabolic stress has not been fully elucidated. Krüppel-associated box-associated protein-1 (KAP1), also known as TRIM28, is a transcriptional co-regulator participating in multiple cellular physiological processes. The post-translational modifications of KAP1 play an essential role in regulating its function. We have previously demonstrated that KAP1 mRNA is highly expressed in concomitant with the upregulation of genes involving in glycolysis and oxidative phosphorylation in breast cancer. Furthermore, KAP1 is phosphorylated at Serine 473 (pS473) upon the induction of nutrient stresses via reactive oxygen species (ROS)-p38 MAPK signaling in breast cancer cells. The S473 phosphorylation of KAP1 reduces the expression of mitochondrial outer membrane protein mitofusin 2 (MFN2), which prevents the hyperfusion of mitochondria and the overproduction of ROS under nutrient deprivation. However, the mechanism of how KAP1 regulates the expression of MFN2 remains unclear. We have engineered a KAP1-deficient MDA-MB-231 cell line and re-expressed KAP1 S473 wild-type (WT), S473A (phosphorylation-defective) or S473D (phosphorylation-mimic) mutants. By analyzing the mRNA expression profiles from these cell lines, several microRNAs (miRNAs) were shown to be upregulated in the KAP1 S473D-expressing cells, which implied that pS473-KAP1 may regulate MFN2 expression through miRNA. Using several miRNA target prediction algorithms, miR-20b was predicted to target the 3’-untranslated region (UTR) of MFN2 mRNA, and the sequence of target site was conserved through evolution. By performing MFN2 3’UTR dual luciferase reporter assays, we have confirmed that MFN2 3’UTRwas targeted by miR-20b and regulated by the phosphorylation status at S473 of KAP1. Also, miR-20b expression was upregulated upon glucose deprivation and both MFN2 mRNA and protein levels declined in breast cancercells overexpressed with miR-20b mimics. In addition, KAP1 S473D, rather than S473A-expressing cells produced more miR-20b under glucose depletion condition and MFN2 expression levels were inversely correlated with miR-20b expression. Moreover, the analysis of The Cancer Genome Atlas (TCGA) breast tumor datasets indicated that there was higher miR-20b but lower MFN2 expression in advanced breast tumors compared with the primary tumors and normal tissues. In conclusion, these results suggested that the induction of KAP1 S473 phosphorylation under nutrition deprivation inhibited MFN2 by upregulating miR-20b, which prevented mitochondrial hyperfusion and consequently benefited breast cancer cell survival in response to metabolic stress. Citation Format: Yu-Shu Liu, Chun-Ting Cheng, Ching Ouyang, Ge-Jyun Liu, David Kong Ann, Ching-Ying Kuo. Phosphorylation of KAP1 Serine 473 regulates mitochondrial dynamics through miR-20b under metabolic stress in 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 P1-05-03.

Breast cancer is one of the most common cancers and leading causes of death in the women worldwide. The evidence shows efficacy of apatinib against breast cancer. Accordingly, the present study was conducted to investigate the effect of apatinib on apoptosis, cell cycle, and Mitogen‑Activated Protein Kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways in the breast cancer MDA-MB-231 cell line. The effects of apatinib on viability, morphology, tumor spheroid, cell cycle, migration, invasion, and apoptosis of MDA-MB-231 breast cancer cells were evaluated in vitro. In addition, expression of proteins involved in NF-κB and MAPK signaling pathways was evaluated using the western blotting analysis. Apatinib decreased viability, tumor spheroid, migration, and invasion of MDA-MB-231 cells. Furthermore, apatinib altered morphology and regulated cell cycle which followed by apoptosis induction in MDA-MB-231 cells. Apatinib decreased expression of p-p65 and p65 proteins in NF-κB signaling pathways and increased expression of p38, p-p38, JNK, and p-JNK in MAPK signaling pathways. The results suggested that apatinib can inhibit proliferation, migration and invasion of breast cancer cell line MDA-MB-231 through inducing apoptosis, cell cycle arrest, and regulating NF-κB and MAPK signaling pathways.

Deregulation of the basic helix‐loop‐helix family member e41 (BHLHE41) has been characterized as a marker of progression of several cancers. In this study, we aimed to explore the mechanism by which BHLHE41 regulates the invasion of breast cancer cells. BHLHE41 suppresses, whereas the silencing of BHLHE41 promotes tumour invasion of both MCF‐7 and MDA‐MB‐231 cells. Meanwhile, BHLHE41 down‐regulated the transcription and translation of SNAI1, SNAI2, VIM and CDH2, and up‐regulated those of CLDN1, CLDN4 and CDH1. Reporter assay indicated that silencing of BHLHE41 dramatically activated the MAPK/JNK signalling pathway in MCF‐7 cell line and the hypoxia signalling pathway in MDA‐MB‐231 cell line. Furthermore, silencing of BHLHE41 activated the MAPK/JNK signalling pathway by up‐regulating phosphorylated JNK and failed to affect the expression of HIF‐1 alpha in MCF‐7 cells. After blocking the MAPK/JNK signalling pathway by specific inhibitor SP600125, silencing of BHLHE41 failed to promote tumour cell invasion. These results suggest that BHLHE41 facilitates MCF‐7 cell invasion mainly via the activation of MAPK/JNK signalling pathway. In conclusion, although BHLHE41 suppresses tumour invasion in MCF‐7 and MDA‐MB‐231 cell lines, the specific regulatory mechanisms may be different.

© 2022 Elsevier B.V.In this study, a series of new Schiff bases, based on a pyrimidine core, were synthesized by the condensation reactions of 1-amino-5-(4-methylbenzoyl)-4-p-tolylpyrimidin-2(1H)-one (1a) and 1-amino-5-(4-methylbenzoyl)-4-p-tolylpyrimidine-2(1H)-thione (1b) with different aromatic aldehyde derivatives using p-toluene sulfonic acid as a catalyst at refluxing in ethyl alcohol. The structures of these newly synthesized Schiff bases (2-11) were verified by 1H NMR, 13C NMR, IR, LC-MS (only for 2, 3, 5, 6, 8, 9, 11), and elemental analysis. The presence of characteristic peaks proving the formation of imine supports the molecules' structures. The cytotoxic effects of molecules 2-11 on the viability of breast (MDA-MB-231) and colon (DLD-1) cancer cells were investigated using the MTT method. The molecules (2-11) demonstrated in vitro antiproliferative activity in the MDA-MB-231 cell line with IC50 values ranging from 58.59 µM to ˃200 µM. A compound 3 was particularly found to be the most potent anticancer drug candidate in this series with an IC50 value of 58.59 µM in MDA-MB-231. Unfortunately, neither the activity of this compound nor the others are high enough to be compared with the positive control drug cisplatin against MDA-MB-231 and DLD-1. In addition, Density Functional Theory (DFT) calculations of compounds 3, 4 and 8 were performed and the molecular docking studies of these compounds with breast cancer protein, PDB ID: 1JNX were presented to compare with experimental and theoretical results.

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer.

The cancer treatment by laser-conjugated nanomaterial has become a new developing trend due of their unique physicochemical performance. The previous few studies reported the preparation of undoped CuS nanoprisms. The current research was concerned with the Mn doping effect on the CuS nanoprisms and its activity in tumor toxicity of M.D. Anderson-Metastatic Breast 231 (MDA-MB-231) cell line with laser treatment. To prepare a novel CuS and Mn-doped CuS nanoprisms with high surface area by two-phase colloidal method, copper nitrate and sulfur powder were used as sources of copper and sulfur respectively. The prepared nanoprisms were investigated as antibacterial and photothermal agents in MDA-MB-231 cancer treatment using near-infrared (NIR) laser. The Mn-CuS nanoprisms were modified with glutathione (GSH) to decrease the cytotoxicity and increase the biocompatibility. The characterization of synthesized nanoprisms involved the structural, compositional, surface charges, optical, and morphological property analyses. X-ray diffraction (XRD) showed the peaks of hexagonal covellite copper sulfide nanoparticles and additional diffraction peaks at Mn-CuS which are assigned to orthorhombic chalcocite CuS. The transmission electron microscopy (TEM) images showed that the CuS and Mn-CuS nanoparticles have nanoprism morphology. The antibacterial activity test revealed that the activity enhances by doping and the prepared Mn-CuS nanostructures were more effective against the Staphylococcus aureus and Escherichia coli bacteria. The results of photothermal treatment indicated that the cancer cells were effectively killed and the GSH@Mn-CuS nanoprisms are able to be used as an efficient theranostic agent for tumor photothermal therapy in the future.

Leucine zipper putative tumor suppressor 1 (LZTS1) is a tumor suppressor gene located on chromosome 8p22, and the expression is usually decreased in different types of cancers. Here we analyze the correlation between LZTS1 and paclitaxel sensitivity to breast cancer. LZTS1 expression was investigated in MDA-MB-231 cell line by reverse transcriptase-polymerase chain reaction(RT-PCR) and western blot. Cell cycle and apoptosis were detected using flow cytometry. Cell proliferation/viability assays to test drug sensitivity in breast cancer cell line. Human tumor xenograft models to measure chemo-sensitivity in LZTS1 over-expression tumor. The expression of LZTS1 was detected using immunohistochemistry in breast cancer, and the correlation between the LZTS1expression and the effect of chemotherapy was analyzed. In vitro cell proliferation assays revealed that LZTS1 expression was correlated with paclitaxel sensitivity in breast cancer cells. Cell cycle analysis and apoptosis experiments have demonstrated that LZTS1 could enhance the inhibitory effect of paclitaxel on the breast cancer cell cycle and the ability to induce apoptosis. Overexpression of LZTS1 could sensitize human breast carcinoma xenografts to paclitaxel. In addition, tumor with high LZTS1 expression was more sensitive to chemotherapy with paclitaxel in breast cancer. For the first time, we evaluated the relationship between LZTS1 expression and chemotherapy sensitivity using the Collagen gel droplet drug sensitivity test(CD-DST) in primary cell culture of breast cancer. LZTS1 may play an important role in improving the sensitivity of paclitaxel-based breast cancer chemotherapy.

Garlic has been used as a medicinal plant since ancient times and is well-documented to produce several organosulfur compounds (OSCs) that show promising chemopreventative and therapeutic properties. The vinyl disulfide sulfoxide garlic rearrangement product, ajoene, is one of the phytochemicals in garlic that possesses a broad spectrum of biological activities against a variety of cancers both in vitro and in vivo. Our group's previous investigations into ajoene's cytotoxicity have shown that it modifies proteins by S-thioallylating redox-sensitive cysteine residues through a thioldisulfide exchange reaction. Further investigations into this exchange have been the primary aim of this PhD thesis using a biotinylation protocol for trapping out the said protein targets of ajoene to link them to anticancer signalling pathways. The thesis begins in Chapter One with an overview of the chemopreventative and antitumour activity of OSCs from garlic in which a particular emphasis is placed on OSC structure and mechanistic aspects of their cancer biology. Chapter Two discusses the various aspects of ajoene as the target natural product of the thesis. These include its biosynthesis and synthesis, in both native form and as derivatives, for SAR studies, for which a UCT synthesis is described. Mechanistic aspects of ajoene's cytotoxicity towards cancer cells in terms of S-thioallylation via disulfide exchange are then discussed at length. Of crucial importance for the chemical biology studies to follow was the conclusion that S-thioallylation by ajoene is regioselective at the vinyl sulfur, as well as effectively irreversible with suitably reactive cysteine thiol groups. Chapter Three presents an in-depth Chapter on the metabolism and pharmacokinetic properties of ajoene and selected derivatives in a murine xenograft model for human oesophageal cancer (WHCO1). This concluded, disappointingly, that no significant differences in terms of tumour volume, mass and growth rate were observed compared to an untreated control. A follow-up study using a small library of eight ajoene derivatives varying ajoene's different functional groups in a blood stability study, revealed a proportional relationship between the in vitro half-life in blood and the IC50 value for WHCO1 cancer cells. This led to mass spectrometry studies showing that ajoene Sthioallylates the βCys-93 residues in haemoglobin, a chemical modification that most likely explains both ajoene's blood instability and its lack of antitumour activity in vivo. Chapter Four describes the organic synthesis and characterisation of four biotin-ajoene chemical biology probes for chemical biology investigation of S-thiolation in which the background theory of activity-based protein profiling and biotin affinity purification is presented. In the synthesis of these probes, chemical stability emerged as a major stumbling block. Gratifyingly, after several trials, the fourth probe designed and constructed, using a simplified tether involving a convergent “Click”-strategy, turned out to be chemically stable. Subsequent biological validation studies confirmed that the probe retained cytotoxicity against cancer cell lines in vitro (human epithelial mammary gland adenocarcinoma cancer cells (MDA-MB-231) and WHCO1) and shared the same compound–target interaction as its parent ajoene; namely, the regioselective S-thiolation of cellular proteins. Chapter Five constitutes a proteomics study into ajoene's primary protein targets in the MDA-MB-231 cell line using our biotin-ajoene probe. Streptavidin-coated magnetic beads in conjunction with an affinity purification mass spectrometry protocol allowed the isolation and identification of 633 protein targets for ajoene in the MDA-MB-231 proteome. Pathway analyses revealed that ajoene interacts with several targets involved in the control of cell cycles (G2/M cell cycle checkpoint), energy metabolism (glycolysis and pentose phosphate pathway) and the regulation of protein metabolism (translation, folding, quality control and degradation), which supports previously reported cytotoxic modes of action for ajoene against MDA-MB-231 cancer cells. Importantly, we have validated that ajoene S-thioallylates glutathione S-transferase (GSTP1), which is a known cancer-therapy target in breast cancer. Overall, this study complements findings on the cancer-cell protein targets of allicin from crushed garlic, by identifying proteins regulating apoptotic and antiproliferative signalling pathways. These findings support the hypothesis that the anticancer activity of ajoene is due to its S-thioallylation of proteins essential to cellular functions. The thesis concludes with comprehensive experimental and reference sections.

We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) with excellent potency, specificity and drug-like properties was selected for further evaluation. Examination of TR-107 effects in triple-negative breast cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. The antitumor response to TR-107 was investigated using human TNBC MDA-MB-231 cell line-induced xenograft tumors. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

Sarcoplasmic reticulum CaTechniques such as real-time polymerase chain reaction, optical microscopy, proliferation and cell death assays, as well as bioinformatic analyses were used.To evaluate the expression levels of the ATP2A2 and ATP2A3 genes in cell lines representative of different subtypes of breast cancer.The results show that the MDA-MB-231 cell line expresses lower mRNA levels for the ATP2A3 gene in comparison with MCF-7 cells. The use of the phytoestrogen resveratrol induces ATP2A3 expression, decreases proliferation, and induces apoptosis in both cell types.SERCA expression might function as a tool to differentiate breast cancer subtypes, which have different treatment requirements.Las enzimas SERCA son esenciales para la homeostasis intracelular de CaSe utilizaron estudios como reacción en cadena de la polimerasa en tiempo real, microscopia óptica, ensayos de proliferación y muerte celular, así como análisis bioinformáticos.Evaluar los niveles de expresión de los genes ATP2A2 y ATP2A3 en líneas celulares que representan diferentes subtipos de cáncer de mama.La línea celular MDA-MB-231 expresa niveles más bajos del ARNm para el gen ATP2A3, en comparación con las células MCF-7. El uso del fitoestrógeno resveratrol induce la expresión de ATP2A3, disminuye la proliferación e induce apoptosis en ambos tipos de células.La expresión de SERCA puede funcionar como una herramienta para diferenciar los subtipos de cáncer de mama, los cuales tienen distintas necesidades de tratamiento.

Cannabis has been used for various medicinal applications including, but not limited to, cancer: most commonly to treat chemotherapy-associated side effects. Cannabis is often used for its palliative effects in the form of purified cannabinoids, or as extracts. This study was conducted using two breast cancer cell lines and aimed to evaluate potential anti-proliferative “intra-entourage effects” between purified phytocannabinoids resembling the THC and CBD ratios of medicinal and recreational cannabis strains, as well as to investigate potential “inter-entourage effects” between the different ratios and the phytochemicals found in a Cannabis sativa extract. This study also aimed to evaluate the potential interaction between cannabinoids and chemotherapeutic agents. The data identified an intra-entourage effect present in the MCF-7 cells when treated with a recreational, but not a medicinal, cannabis formulation. This effect may be due to THC partially exerting its anti-proliferative effects through the estrogen receptor (ER), present in the MCF-7 cell line. Little to no intra-entourage effects were observed in the MDA-MB-231 cell line and no inter-entourage effects were observed in either cell line. The simultaneous treatment of the MCF-7 cell line with various cannabinoid formulations and the common breast cancer treatment, tamoxifen, resulted in the diminished anti-proliferative activity of tamoxifen, an effect that was more evident when combined with recreational cannabis formulations. Since cannabis is commonly used in palliative care to treat chemotherapy-associated side effects, further research is required to investigate the potential interference of various cannabis formulations to ensure that the efficacy of chemotherapeutic agents is not compromised. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03102-1.

The development of dual-stimuli-responsive hydrogels attracts much research interest owing to its unique stimuli-responsive characteristics. In this study, a poly-N-isopropyl acrylamide-co-glycidyl methacrylate-based copolymer was synthesized by incorporating N-isopropyl acrylamide (NIPAm) and a glycidyl methacrylate (GMA) monomer. The synthesized copolymer, pNIPAm-co-GMA was further modified with L-lysine (Lys) functional units and further conjugated with fluorescent isothiocyanate (FITC) to produce a fluorescent copolymer pNIPAAm-co-GMA-Lys hydrogel (HG). The in vitro drug loading and dual pH- and temperature-stimuli-responsive drug release behavior of the pNIPAAm-co-GMA-Lys HG was investigated at different pH (pH 7.4, 6.2, and 4.0) and temperature (25 °C, 37 °C, and 45 °C) conditions, respectively, using curcumin (Cur) as a model anticancer drug. The Cur drug-loaded pNIPAAm-co-GMA-Lys/Cur HG showed a relatively slow drug release behavior at a physiological pH (pH 7.4) and low temperature (25 °C) condition, whereas enhanced drug release was achieved at acidic pH (pH 6.2 and 4.0) and higher temperature (37 °C and 45 °C) conditions. Furthermore, the in vitro biocompatibility and intracellular fluorescence imaging were examined using the MDA-MB-231 cell line. Therefore, we demonstrate that the synthesized pNIPAAm-co-GMA-Lys HG system with temperature- and pH-stimuli-responsive features could be promising for various applications in biomedical fields, including drug delivery, gene delivery, tissue engineering, diagnosis, antibacterial/antifouling material, and implantable devices.

Most mortality associated with breast cancer (BC) is caused by metastasis. The metastatic process involves dissemination of tumor cells from the primary site to distant organs through the bloodstream. The tumor cells found in the bloodstream are called circulating tumor cells (CTCs). The number of CTCs is an independent predictor of poor survival in BC patients. We and others have found that CTCs exist as single cells or in clusters. CTC clusters have up to 100 times more metastatic potential, higher cell survival, and more resistance to chemotherapy than single CTCs in BC, regardless of the subtype or prior treatment. Our published transcriptomic and proteomic studies have recently identified high B-cell lymphoma 2 (Bcl2) expression and downregulation of apoptosis pathways in triple-negative BC tumors associated with CTC clusters using patient-derived xenograft (PDX) models. Bcl2 is an oncogene that promotes survival and regulates pro- and anti-apoptotic pathways in cancer. Approximately 75% of breast tumors are positive for Bcl2. Also, the expression of Bcl2 is a poor prognostic marker in TNBC patients, mainly in the absence of adjuvant therapy. However, the exact role of Bcl2 in clustering of cells needs further investigation. In the current study, we aimed to evaluate concentration-dependent effects of the Bcl2 inhibitor, venetoclax, on cellular growth in TNBC cells. We first evaluated the cell growth patterns over time of two different cell numbers plated per well. We also generated 8-point concentration-response curves (10nM - 30μM) of venetoclax in MDA-MB-231 cell line to assess cell growth. The cells were plated in 96-well plates overnight followed by the addition of venetoclax and daily image analysis to obtain cell count using EnSight multimode plate reader. We found that 500 cells per well allowed us to obtain cell growth data for up to 6 days and 2,000 cells per well for up to 3 days. Therefore, 500 cells per well were selected to determine the effects of venetoclax over a 6-day period. On day 6, venetoclax treatment inhibited cell growth in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) of venetoclax was 40 μM. Next, we evaluated the effects of venetoclax on disruption of cellular clustering in MDA-MB-231 cells. We plated 10,000 cells per well in 6-well plates and incubated for overnight attachment. The cells were treated with venetoclax (10μM and 30μM) and trypsinized for a short-time (one minute) after 72 hours of treatment. The number of clusters with 3 or more cells clumped together were counted in 5 independent images using Image J. We found that there was a significant decrease in the number of clusters with venetoclax at 30μM. Our ongoing studies focus on exploring the effects of Bcl2 knockdown by siRNAs on cell growth and on disruption of cellular clustering in TNBC cells. Citation Format: Alexis Jordan Ruiz, Nicolas Park, Noor Abdulkareem, Meghna Trivedi. Evaluating the effects of Bcl2 inhibitor, venetoclax, on cellular growth and clustering in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2477.

After lung cancer, breast cancer is the second leading cause of cancer death in women in the United States, accounting for 12.5 % of all new cancer cases globally. Triple-negative breast cancer (TNBC) remains the hardest breast cancer and is difficult to treat. The current treatment option for TNBC is the surgical removal of the tumor, followed by chemotherapy. However, patients with advanced diseases respond poorly to current treatments. Therefore, discovering more effective and less toxic small drug molecules with a unique mechanism of action is one of the top priorities in drug discovery programs. Chromones have a benzo-pyrone skeleton and display interesting pharmacological activities, such as anti-cancer, anti-HIV, antiviral and anti-inflammatory, with low toxicity. In continuation of our current research work, we report the design and synthesis of substituted chromone-2-carboxamides by attaching substituted phenyl, tetrahydropyridine, and tetrahydroisoquinoline groups as anti-breast cancer agents. The starting material, substituted chromone-2-carbonyl chloride, was obtained by the acylation reaction of corresponding carboxylic acids using phosphorous pentachloride in dry cyclohexane. An equimolar amount of acyl chloride was slowly added to a stirred solution of corresponding substituted N-amino salt in dry THF in the presence of triethyl amine and refluxed at 70 °C that led to the formation of the ylides. The ylides were reduced using sodium borohydride to yield the desired substituted chromone-2-carboxamides in moderate to good yields. The synthesized compounds were characterized by NMR and elemental analysis. These compounds were evaluated for their cytotoxic effects on MBA-MB-231 ER-ve breast cancer cell lines using a Synergy HTX multi-mode reader (Bio-Tek, Winooski, VT, USA) with excitation/emission wavelength settings at 550/580. Compound MG-KKR-5-140A showed the most potent cytotoxicity with an IC50 value of 0.82 µg/mL on the MDA-MB-231 cell line. In-silico pharmacophore hypotheses were generated using GALAHAD and PHASE, and the best models with probable bioactive conformation(s) for these compounds were proposed. These confirmations and the alignments of the molecular structures give us an insight into designing compounds with better biological activity. This research was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54 MD007582. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Suresh Eyunni, Elizabeth Mazzio, Karam F. A. Soliman. Antiproliferative activity of substituted chromone-2-carboxamides on MDA-MB-231 cells and their 3D pharmacophore models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3834.

Herein, we investigate the combinatorial therapeutic effects of naturally occurring flavonoids kaempferol (K) and fisetin (F) on triple-negative breast cancer (TNBC: MDA-MB-231 cell line). Dose-dependent MTT assay results show that K and F exhibited cytotoxicity in MDA-MB-231 cells at 62 and 75 μM (IC50), respectively, after 24 h. However, combined K + F led to 40% and more than 50% TNBC cell death observed at 10 and 20 μM, respectively, which revealed the synergistic association of both. The combination of K and F was determined to be more effective in inhibiting cell viability than either of the agents alone. The morphological changes associated with significant apoptotic cell death were observed under a fluorescent microscope, strongly supporting the synergistic association between K and F. We also proposed that combining the effects of both polyphenols, as opposed to their individual effects, would increase their in vitro efficacy. Furthermore, we assessed the cell death pathway by the combinational treatment via reactive oxygen species-induced DNA damage and the mitochondrially mediated apoptotic pathway. This study reveals the prominent synergistic role of phytochemicals, which helps in elevating the therapeutic efficacy of dietary nutrients and that anticancer effects may be a result of nutrients that act in concert.

Triple-negative breast cancer (TNBC) represents an aggressive subtype of breast cancer (BC) with a typically poorer prognosis than other subtypes of BC and limited therapeutic options. Therefore, new drugs would be particularly welcome to help treat TNBC. Preussin, isolated from the marine sponge-associated fungus, Aspergillus candidus, has shown the potential to reduce cell viability and proliferation as well as to induce cell death and cell cycle arrest in 2D cell culture models. However, studies that better mimic the tumors in vivo, such as 3D cell cultures, are needed. Here, we studied the effects of preussin in the MDA-MB-231 cell line, comparing 2D and 3D cell cultures, using ultrastructural analysis and the MTT, BrdU, annexin V-PI, comet (alkaline and FPG modified versions), and wound healing assays. Preussin was found to decrease cell viability, both in 2D and 3D cell cultures, in a dose-dependent manner, impair cell proliferation, and induce cell death, therefore excluding the hypothesis of genotoxic properties. The cellular impacts were reflected by ultrastructural alterations in both cell culture models. Preussin also significantly inhibited the migration of MDA-MB-231 cells. The new data expanded the knowledge on preussin actions while supporting other studies, highlighting its potential as a molecule or scaffold for the development of new anticancer drugs against TNBC.

Over the years, carbazoles have been largely studied for their numerous biological properties, including antibacterial, antimalarial, antioxidant, antidiabetic, neuroprotective, anticancer, and many more. Some of them have gained great interest for their anticancer activity in breast cancer due to their capability in inhibiting essential DNA-dependent enzymes, namely topoisomerases I and II. With this in mind, we studied the anticancer activity of a series of carbazole derivatives against two breast cancer cell lines, namely the triple negative MDA-MB-231 and MCF-7 cells. Compounds 3 and 4 were found to be the most active towards the MDA-MB-231 cell line without interfering with the normal counterpart. Using docking simulations, we assessed the ability of these carbazole derivatives to bind human topoisomerases I and II and actin. In vitro specific assays confirmed that the lead compounds selectively inhibited the human topoisomerase I and interfered with the normal organization of the actin system, triggering apoptosis as a final effect. Thus, compounds 3 and 4 are strong candidates for further drug development in multi-targeted therapy for the treatment of triple negative breast cancer, for which safe therapeutic regimens are not yet available.

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that belongs to the serine protease inhibitor (serpin) family. An increase in PEDF activity has been shown to be a potent inhibitor of tumour progression and proliferation, suggesting a possible therapeutic target. There is still a great deal to learn about how PEDF controls metabolic pathways in breast cancer and its metastatic form. Given this, the primary purpose of this study was to use a metabolomics approach to gain a better understanding of the mechanisms driving the reprogramming of metabolic events involved in breast cancer pertaining to PEDF under various glycaemic loads. We employed gas chromatography–quadrupole mass spectrometry (GC-Q-MS) to investigate metabolic changes in the triple-negative breast cancer (TNBC) cell line MDA-MB-231 treated with PEDF under glycaemic loading. Multivariate and univariate analyses were carried out as indicative tools via MetaboAnalyst (V.5.0) and R packages to identify the significantly altered metabolites in the MDA-MB-231 cell line after PEDF exposure under glycaemic loading. A total of 61 metabolites were found, of which nine were selected to be distinctively expressed in MDA-MB-231 cells under glycaemic conditions and exhibited differential responses to PEDF (p < 0.05, VIP > 1). Abnormalities in amino acid metabolism pathways were observed. In particular, glutamic acid, glutamine, and phenylalanine showed different levels of expression across different treatment groups. The lactate and glucose-6-phosphate production significantly increased in high-glucose vs. normal conditions while it decreased when the cells were exposed to PEDF, confirming the positive influence on the Warburg effect. The TCA cycle intermediates, including malate and citric acid, showed different patterns of expression. This is an important finding in understanding the link of PEDF with metabolic perturbation in TNBC cells in response to glycaemic conditions. Our findings suggest that PEDF significantly influenced the Warburg effect (as evidenced by the significantly lower levels of lactate), one of the well-known metabolic reprogramming pathways in cancer cells that may be responsive to metabolic-targeted therapeutic strategies. Moreover, our results demonstrated that GC-MS-based metabolomics is an effective tool for identifying metabolic changes in breast cancer cells after glycaemic stress or in response to PEDF treatment.

Even though Olea europaea L. is one of the most important and well-studied crops in the world, embryonic parts of the plants remain largely understudied. In this study, comprehensive phytochemical profiling of olive vegetative buds of two Croatian cultivars, Lastovka and Oblica, was performed with an analysis of essential oils and methanol extracts as well as biological activities (antioxidant, antimicrobial, and cytotoxic activities). A total of 113 different volatiles were identified in essential oils with hydrocarbons accounting for up to 60.30% and (Z)-3-heptadecene being the most abundant compound. Oleacein, oleuropein, and 3-hydroxytyrosol had the highest concentrations of all phenolics in the bud extracts. Other major compounds belong to the chemical classes of sugars, fatty acids, and triterpenoid acids. Antioxidant, antimicrobial, and cytotoxic activities were determined for both cultivars. Apart from antioxidant activity, essential oils had a weak overall biological effect. The extract from cultivar Lastovka showed much better antioxidant activity than both isolates with both methods (with an oxygen radical absorbance capacity value of 1835.42 μM TE/g and DPPH IC50 of 0.274 mg/mL), as well as antimicrobial activity with the best results against Listeria monocytogenes. The human breast adenocarcinoma MDA-MB-231 cell line showed the best response for cultivar Lastovka bud extract (IC50 = 150 μg/mL) among three human cancer cell lines tested. These results demonstrate great chemical and biological potential that is hidden in olive buds and the need to increase research in the area of embryonic parts of plants.

Triple-negative breast cancer (TNBC) represents 15-25% of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting. The lipid core of the nanovehicle was composed of a Carnaúba wax matrix that simultaneously incorporated iron oxide nanoparticles and doxorubicin (DOX) - a chemotherapeutic drug. These drug-loaded wax nanovehicles were modified with a combination of two functional and complementary molecules: (i) a mannose ligand (macrophage targeting) and (ii) an acid-sensitive sheddable polyethylene glycol (PEG) moiety (specificity). The TAMs targeting strategy relied on the mannose-mannose receptor recognition exclusively after acid-sensitive "shedding" of the PEG in the relatively low tumour microenvironment pH. The pH-induced targeting capability towards TAMs was confirmed in vitro in a J774A.1 macrophage cell line at different pH (7.4 and 6.5). Biocompatibility and efficacy of the final targeted formulations were demonstrated in vitro in the TNBC MDA-MB-231 cell line and in vivo in an M-Wnt tumour-bearing (TNBC) mouse model. A preferential accumulation of the DOX-loaded lipid nanovehicles in the tumours of M-Wnt-tumour bearing mice was observed, which resulted both on an efficient tumour growth inhibition and a significantly reduced off-target toxicity compared to free DOX. Additionally, the developed magnetic hybrid nanovehicles showed outstanding performances as T