MDA-MB231 Cells Research Articles

Phytochemical-rich extracts of Helianthemum lippii possess antimicrobial, anticancer, and anti-biofilm activities

Helianthemum lippii is a perennial shrubby plant growing in the sandy environments of Italy, Mediterranean countries of North Africa and Middle East. H. lippii is used in traditional medicine but there are very few reports referring to the phytochemical characterization, the ethnopharmacology, and the biological activity of H. lippii. The goal of this study was to determine the phytochemical composition of different H. lippii extracts, cold (CME) and hot (HME) methanol, cold (CPEE) and hot (HPEE) petroleum ether, as well as to evaluate their anticancer and antimicrobial activities and biofilm formation reduction. Fifty-fours phytocompounds have been determined by HPLC-UV-ESI-QTOF-MS analysis. All the four extracts reduced the viability of human MDA-MB231 and HCT116 cells, being cold and hot methanol extracts the most effective. The antimicrobial activity against S. aureus, P. aeruginosa, E. faecalis, E. coli, C. albicans was also evaluated. Data showed the greater susceptibility of S. aureus to hot methanolic extract. Concerning antifungal activity, C. albicans resulted more susceptible to petroleum ether. Moreover, some of the samples exhibited a good antibiofilm activity both on immature and on mature biofilms. The four extracts showed interesting antimicrobial and cytotoxic activities and can be considered good candidates for new therapeutic applications

Chemical composition, cytotoxic effects, antimicrobial and antibiofilm activity of Artemisia arborescens (Vaill.) L. growing wild in the province of Agrigento, Sicily, Italy

Artemisia arborescens (Vaill.) L. is a perennial shrubby plant growing along the coastal strips of the Mediterranean region. It is used in traditional medicine. Its essential oil and solvent extracts exhibit a very interesting chemotherapeutic potential, which makes this plant useful in maintaining human health. The goal of this study was to determine the phytochemical composition of the petroleum ether and methanol extracts, as well as to evaluate anticancer activities and antimicrobial and biofilm formation reduction. Thirty-nine phytochemical compounds in negative ion mode and 25 in positive ion mode were identified by HPLC-ESI-QTOF-MS. All four extracts reduced the viability of human MDA-MB231 and HCT116 cancer cells suggesting a similar cytotoxic efficacy of the different extracts in MDA-MB231 and more pronounced antiproliferative effects on HCT116 cells treated with HPEE and CPEE. Antimicrobial activity was exhibited by the hot petroleum ether extract against all tested bacteria (MIC 0.15–2.5 mg/mL), except for S. agalactiae. Both methanol extracts showed activity against C. albicans with a MIC value of 2.5 mg/mL, and a lack of antibacterial activity. To whom concern the anti-biofilm activity, hot methanol extract shows the highest activity in inhibiting biofilm formation among all extract.

Pharmacotranscriptomic profiling of resistant triple-negative breast cancer cells treated with lapatinib and berberine shows upregulation of PI3K/Akt signaling under cytotoxic stress.

Triple-negative breast cancer (TNBC) is the most incurable type of breast cancer, accounting for 15-20% of breast cancer cases. Lapatinib is a dual tyrosine kinase inhibitor targeting EGFR and Her2, and berberine (BBR) is a plant-based alkaloid suggested to inhibit several cancer signaling pathways. We previously reported that lapatinib activates the Akt oncoprotein in MDA-MB231 TNBC cells. The present study determined the mechanism(s) of Akt activation in response to lapatinib, BBR, and capivasertib (Akt inhibitor) as well as the role of Akt signaling in chemoresistance in TNBC cells. Genetic profiles of 10 TNBC cell lines and patients were analyzed using datasets obtained from Gene Expression Omnibus and The Cancer Genome Atlas Database. Then, the effects of lapatinib, BBR, and capivasertib on treated MDA-MB231 and MCF-7 cell lines were studied using cytotoxicity, immunoblot, and RNA-sequencing analyses. For further confirmation, we also performed real-time PCR for genes associated with PI3K signaling. MDA-MB231 and MCF-7 cell lines were both strongly resistant to capivasertib largely due to significant Akt activation in both breast cancer cell lines, while lapatinib and BBR only enhanced Akt signaling in MDA-MB231 cells. Next-generation sequencing, functional enrichment analysis, and immunoblot revealed downregulation of CDK6 and DNMT1 in response to lapatinib and BBR lead to a decrease in cell proliferation. Expression of placental, fibroblast growth factor, and angiogenic biomarker genes, which are significantly associated with Akt activation and/or dormancy in breast cancer cells, was significantly upregulated in TNBC cells treated with lapatinib and BBR. Lapatinib and BBR activate Akt through upregulation of alternative signaling, which lead to chemoresistance in TNBC cell. In addition, lapatinib overexpresses genes related to PI3K signaling in resistant TNBC cell model.

Combinatorial effects of telmisartan and docetaxel on cell viability and metastatic gene expression in human prostate and breast cancer cells.

The epithelial-to-mesenchymal transition (EMT) is a unique process resulting in enhanced cell motility, invasiveness, and metastasis in cancer. The EMT is regulated by several transcription factors, including Snail and Slug, which exert crucial roles during cancer progression. We have studied the effects of Docetaxel as the first-line chemotherapy agent for prostate cancer, and Telmisartan as an anti-hypertensive drug on the expression level of Snail and Slug. In addition, the effects of Docetaxel, Telmisartan and their combination on cancer cell proliferation were investigated. The PC3, DU145, MDA-MB468, and HEK cell lines were used for this study. Quantitative RT-PCR analysis and MTT assay were used to study the expression of Snail and Slug level and cell proliferative assay, respectively. We found that a combination of Docetaxel + Telmisartan effectively inhibits the cell proliferation in cancerous cells in comparison with each drug alone (P<0.05). Furthermore, in these cell lines, Docetaxel, Telmisartan and their combination significantly diminished the expression level of Snail and Slug genes compared to control cells (P<0.001), however, in the HEK cell line, this effect was seen only in the combination group. Our data imply that Telmisartan and its combination with Docetaxel exert strong inhibitory effects on the expression level of Snail and Slug genes. Also, these drugs and their combination could inhibit cancer cell proliferation. In conclusion, the combination of Telmisartan and Docetaxel has the potential to suppress the metastasis of prostate and breast cancer cells.

51P Mitochondrial pro-apoptotic properties of isolated compounds from two soft corals of Persian Gulf

Soft corals are an important source of cytotoxic organisms. Some locals use these marine organisms traditionally to treat cancer. Therefore, this study was designed to isolate the cytotoxic extracts and compounds of two species of sinularia soft corals along with their mechanisms against MCF-7 and MDA-MB231 cell lines through MTT assay, Annexin PI flow cytometry, generation of ROS, and also mitochondrial membrane potential.

Chemical alternative for cell identification and cross-contamination detection.

Misidentification of human cell lines has previously led to confusing results during cell culture experiments. Although several enzymatic as well as molecular analysis approaches have been developed for cell-line authentication, these methods remain costly. In the present paper, we describe a simple chemical alternative based on known compound cell cytotoxicity. In addition to cisplatin, a pool of eight tamoxifen derivative compounds was used to compare the cytotoxic effects on three different breast cancer cell lines: MCF-7, T47D and MDA-MB-231. Our results show that four out of the eight cytotoxic-related compounds allowed to distinguish the different cell lines based on their IC50 (the half maximal inhibitory concentration) values which are cell type dependent. The remaining chemicals, particularly the most cytotoxic P15, showed close IC50 values for all the cell lines. Interestingly, flow cytometry experiments have identified notable differences among the three cell lines treated with P15. T47D and MDA-MB231 cells were blocked in SubG1 phase and S phase, respectively, while no significant change in cell cycle profile was noticed for MCF-7 cells. Differences were also noted at the level of caspase-3 activity and cell proliferation in P15-treated cells.

Abstract P5-10-03: Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome

Abstract TP53 is the most frequently mutated gene in most types of human cancer, including breast cancer. The triple-negative breast cancer (TNBC) subtype in particular displays TP53 mutation in approximately 80% of patients. Unlike other breast cancer subtypes (e.g., ER/PR-positive or HER2-positive), TNBC patients currently lack an approved highly effective targeted therapy. Notably, most TNBC acquires TP53 mutations, which, in addition to the loss of canonical p53 functions, can result in gain-of-function, activating different cellular mechanisms involved in tumor phenotypes such as proliferation, metastasis, invasiveness, and angiogenesis. Here we evaluate the role of mutant p53 in cancer phenotypes of TNBC cell lines, in particular their inflammatory profile. As expected, loss of p53 protein by small interfering RNA depletion did not show a major impact on the cell viability of MDA-MB231 and Hs578t cells in MTT assay. However, depleting mutant p53 knockdown made MDA-MB231 cells more susceptible to treatment with methyl-methane sulfonate, a genotoxic alkylating agent. Interestingly, cell invasion as measured by the transwell assay demonstrated that depletion of mutant p53 depletion decreased the invasiveness potential of MDA-MB231 and Hs578t cells which was substantiated with decreased migration of MDA-MB231 cells in a scratch assay over 24 h. RNA sequencing of MDA-MB231 and Hs578t cells revealed that mutant p53 knockdown decreased the expression of several constitutively expressed pro-inflammatory cytokines such as IL8, IL6, CXCL2, and CXCL3, but not genes associated with survival to alkylating agents (NRF2 and Endoplasmic reticulum stress markers) or genes typically regulated by wild-type p53 when compared to control-silenced and MMS-treated cells as indicated by Pathway Enrichment Analysis using the Enrich R and DAVID tools. These results were confirmed by ELISA quantification of IL8, IL6, and CXCL2 in MDA-MB231 and Hs578t transfected with two sequences of siRNA targeting mutant p53. On the other hand, RNA sequencing revealed some constitutively expressed genes known to be involved in breast cancer cells malignancy, such as PTGS2 (COX-2 enzyme gene) and MMP1, which were not affected by p53 knockdown. We found that MMP1, and PGE2 (the product of PTGS2/COX-2 enzyme), are upregulated by the ERK1/2 MAPK signaling pathway as determined in MDA-MB231 cells treated with the MEK1/2 inhibitor UO126 and sorafenib. UO126 and sorafenib also decreased IL8 and IL6 production. Furthermore, combined mutant p53 knockdown with MEK/ERK1/2 pathway inhibition caused a more pronounced IL8 and IL6 inhibition when compared to either p53 knockdown or UO126/sorafenib alone, whereas MMP1 and PGE2 levels were only reduced by MEK/ERK1/2 inhibitor treatments. Interestingly, neither mutant p53 knockdown impacted ERK1/2 phosphorylation status nor did UO126/sorafenib alter mutant p53 immunocontent. Reporter assays showed that mutant p53 promotes NFkappaB reporter activation, and MEK/ERK1/2 controls both NFkappaB and AP-1 transcription factors, both associated with the expression of the secretome components evaluated herein. Functional cell assays showed that concomitant inhibition of mutant p53 and MEK/ERK1/2 pathways reduce cell proliferation, invasion, and migration, indicating that mutant p53 protein gain of function cooperates with ERK1/2 MAPK signaling pathway to promote secretome production and malignant phenotypes in TNBC cell models. Citation Format: Raquel Nascimento das Neves, Aparna Gorthi, Alexander James Roy Bishop, Alfeu Zanotto Filho. Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-03.

Abstract P5-17-01: Targeting Acetyl-CoA carboxylase in pre-clinical breast cancer models

Abstract Background: Cancer cells and normal cells of the same lineage differ in their metabolism. We previously described large scale shifts in isoenzyme distribution between matching cancer and normal tissues and identified Acetyl-coA carboxylase (ACC1/ACACA) as a cancer dominant enzyme that is overexpressed in multiple cancer types. ACC1 catalyzes the initial rate-limiting step in de novo fatty acid synthesis, the conversion of acetyl-CoA to malonyl-CoA. Gene knock-out experiments demonstrated that this enzyme is essential for cancer growth. In this study, we evaluated the in vitro and in vivo efficacy of a small molecule ACC inhibitor, PF05175157 as a potential anticancer drug. This drug has been tested in clinical trials for diabetes, but development was discontinued due to grade 2 thrombocytopenia.Methods: We performed in vitro cytotoxicity assays in 15 breast cancer cell lines and in normal mammary epithelial HMEC cells, examined effect on apoptosis and cell cycle progression, and tested for synergy with alpelisib, docetaxel, doxorubicin, everolimus, iniparib, neratinib and TEPP46 (PKM2 and PKLR activator). We next assessed in vivo single agent activity in a triple negative patient derived (PDX) model (J000102184) in NSGTM mice and in MDAMB468 xenografts implanted into Rag2/IL2RG double knockout mice. We performed RNA sequencing and metabolomic profiling of cells treated with PF05175157 to study metabolic and transcriptomic effects of the drug. Results: PF05175157 induced time and dose dependent growth inhibition in all but 1 of the 15 cancer cell lines. The estimated EC50 after 72h exposure ranged from 0.95 to 76 μg/mL in T47D and BT549 cells, respectively (Cmax of 20 μg/mL can be achieved in human serum). There was no significant inhibitory effect on HMEC cells. In cancer cell lines, the % of apoptotic cells increased from 4% to 8% in BT474 and from 7.7% to 17.8% in MDMBA468 cells upon treatment with the compound, and there was a trend towards G2/M cell cycle arrest in both cell lines after 72 hours of exposure (10μg/mL). In drug combination experiments, PF05175157 added to iniparib, or to the PKM2 activator, TEPP46, decreased cell viability compared to single agent therapies in several cell lines. PF05175157 significantly delayed tumor growth compared to vehicle, when administered orally (20mg/kg gavage BID) in a TNBC PDX model (median tumor volume after 33 days: 334.1 mm3 in PF05175157-treated vs. 490.5 mm3 in methylcellulose-treated mice; p=3.39e-7) and intraperitoneally (20 mg/kg in DMSO) in an MDAMB468 xenograft model (median tumor volume after 40 days: 244.5 mm3 in PF05175157-treated vs. 303.3 mm3 in DMSO-treated mice; p&amp;lt;0.05). Transcriptomic and metabolic profiling of MDAMB468 and BT474 cells treated with 10 ug/ml PF05175157 for 6 and 24 hours revealed activation of immune signaling, epigenetic regulation and DNA damage repair pathways along with down-regulation of a broad range of metabolic pathways. Conclusions: The small molecule ACC inhibitor, PF05175157, has significant single agent in vitro and in vivo growth inhibitory effect on a range of breast cancer cell lines at concentrations that can be achieved in human serum. It showed synergy with iniparib and another metabolic inhibitor (TEPP46). Targeting de novo fatty acid synthesis by inhibiting ACC is a promising therapeutic strategy. Citation Format: Julia Foldi, Michal Marczyk, Vignesh Gunasekharan, Tao Qing, Raghav Sehgal, Naing Lin Shan, Viswanathan Muthusamy, Sheila Umlau, Yulia V. Surovtseva, Richard Kibbey, Lajos Pusztai. Targeting Acetyl-CoA carboxylase in pre-clinical breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-01.

The natural isoflavone Biochanin-A synergizes 5-fluorouracil anticancer activity in vitro and in vivo in Ehrlich solid-phase carcinoma model.

Isoflavones are considered one of the most extensively studied plant-derived phytoestrogenic compounds. Of these, Biochanin A (Bio-A), a natural isoflavone abundant in cabbage, alfalfa, and red clover, has drawn a lot of attention. As reported in multiple studies, Bio-A possesses a promising anticancer activity against estrogen receptor-positive (ER+) breast cancer. The current study investigated the working hypothesis that Bio-A could synergistically enhance the potency of 5-fluorouracil (5-FU) in ER+ breast cancer. The hypothesis was tested both in vitro on hormone receptor-positive (MCF-7) and triple-negative breast cancer cells (MDA-MB231). Additionally, in vivo studies were performed in the Ehrlich solid-phase carcinoma mouse model. The in vitro cytotoxicity studies revealed that Bio-A synergistically increased the potency of 5-FU in both MCF-7 and MDA-MB231 cell lines. The synergistic effect of 5-FU/Bio-A combination was verified in vivo. The combination therapy (where 5-FU was used at one fourth its full dose) led to a significant 75% reduction in tumor volume after two treatment cycles. This was in addition to producing a significant 2.1-fold increase in tumor necrosis area% compared to mock-treated control. In conclusion, the current study presents the first preclinical evidence for the potential merit of 5-FU/Bio-A combination for the treatment of ER+ breast cancer. The synergistic antitumor effect of Bio-A/ 5-FU combination can be, at least partly, attributed to Bio-A-mediated suppression of ER-α/Akt axis and the augmentation of 5-FU-mediated proapoptotic effects. © 2022 John Wiley & Sons, Ltd.

Increased expression of Profilin potentiates chemotherapeutic agent-mediated tumour regression.

Targeted cancer therapy is an alternative to standard chemotherapy for a better prognosis. Although its incompetency for triple-negative breast cancer (TNBC), treatment still relies on classical chemotherapy. Increasing evidence suggest that chemotherapeutic drug-induced toxic effect could be minimised by combinatorial therapy. Profilin's familiar anti-tumorigenic activity can be utilised in combination with the drug to improve efficacy, which could be promising therapeutics to treat TNBC. All-trans retinoic acid (ATRA) in combination with vinblastine was tested on human MDA MB-231 cell line (MB-231) (in vitro) and MB-231 borne breast cancer in nude mice (in vivo). Effects of combination treatment on tumour growth inhibition and apoptosis were examined by tumour volume, histology and PARP cleavage. ATRA-induced transcriptional regulation of profilin had been evaluated by gel-shift and reporter gene assays. Profilin's role in ATRA-induced vinblastine efficacy was validated in profilin-stable and profilin-silenced cells. ATRA binds with RAR/RXR to increase the profilin expression that potentiated cell death by chemotherapeutics. ATRA priming led to vinblastine-mediated potentiation of G2-M phase cell cycle arrest in MB-231 cells and regression of breast cancer in xenograft mice at very low concentration without any adverse effects. Moreover, increased p53 and PTEN but downregulated p65 in the tumour tissues further supported the involvement of profilin for tumour regression. Vinblastine at very low concentration (20 times lesser than the recommended dose for breast cancer therapeutic) significantly regress tumour growth in ATRA-primed mice without any toxic effects suggesting potential combinatorial therapeutics for TNBC.

Synthesis and Biological Activity of 3-(Heteroaryl)quinolin-2(1H)-ones Bis-Heterocycles as Potential Inhibitors of the Protein Folding Machinery Hsp90.

In the context of our SAR study concerning 6BrCaQ analogues as C-terminal Hsp90 inhibitors, we designed and synthesized a novel series of 3-(heteroaryl)quinolin-2(1H), of types 3, 4, and 5, as a novel class of analogues. A Pd-catalyzed Liebeskind–Srogl cross-coupling was developed as a convenient approach for easy access to complex purine architectures. This series of analogues showed a promising biological effect against MDA-MB231 and PC-3 cancer cell lines. This study led to the identification of the best compounds, 3b (IC50 = 28 µM) and 4e, which induce a significant decrease of CDK-1 client protein and stabilize the levels of Hsp90 and Hsp70 without triggering the HSR response.

In-silico activity prediction, structure-based drug design, molecular docking and pharmacokinetic studies of selected quinazoline derivatives for their antiproliferative activity against triple negative breast cancer (MDA-MB231) cell line

BackgroundCancer is a major health threat especially in unindustrialized nations. It surpasses coronary diseases and takes the number one killer position as a result of different global wide influences. Among many breast cancer substrates, triple-negative breast cancer (TNBC) is particularly devastating because it rapidly metastasize to other parts of the body, with a high risk of earlier recession and mortality.ResultIn this research work, four (4) quantitative structure activity relationship (QSAR) models were developed using a series of quinazoline derivatives with activities against triple negative breast cancer cell line (MDA-MB231), model 1 was selected due to its statistical fitness with the following validation parameters: R2 = 0.875, Q2 = 0.837, R2 − Q2 = 0.038, Next test set = 5, and R2ext = 0.655. Molecular docking studies was performed for the quinazoline series as well as the reference drug (Gefitinib) and the active site of the epidermal growth factor receptor (EGFR) (pdb id = 3ug2). Eight compounds (6, 10, 13, 16, 17, 18, 19 and 20) were observed to have better docking score docking scores relative to Gefitinib. Compound number nineteen from the training set (pred pIC50 = 5.67, Residual = − 0.04 and MolDock score = − 123.238) was identified as the best compound since it has the best Moldock score and was excellently predicted by the selected model with least residual value, Hence was adopted as template for the design of Ten (10) new novel compounds with better activities and better docking scores. The inhibitive activities of the designed compounds were predicted by the selected model and most of them possess an improved activity relative to the template compound (19). The designed compounds were also redocked on to active pocket of the EGFR receptor and it was observed that they displayed better docking scores compared to the Template and the reference drug (Gefitinib) utilized in the design. Furthermore, the designed compounds were subjected to ADMET and drug-likeness studies using SWISSADME and pkCSM online web tools and they were observed to be pharmacologically active, easily synthesized and do not violate the Lipinski’s rule of five.ConclusionHence, the designed compounds can be employed as inhibitors of MDA-MB231 cell line after passing through in vivo and in vitro evaluation.

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

Monoclonal Antibody Against Sortilin Induces Apoptosis in Human Breast Cancer Cells.

Background:Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells.Methods:In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines.Results:Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines.Conclusion:Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.

Anticancer Activity of Elytraria acaulis L. Extracts on Triple Negative Breast Cancer Cell Line

Objectives: In the present study, the anticancer potential of the plant extracts of Elytraria acaulis L. (E. acaulis L.) were investigated on human breast cancer cells (MDAMB-231). Methods: Different organic solvent extras.cts of E. acaulis L. were prepared using methanol, ethanol, ethyl acetate and hexane. The cytotoxicity of plant extracts were evaluated by MTT assay (-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) against MDA MB-231cells. The apoptosis associated morphological changes were also observed with the help of propidium iodide staining method. Results: Of all the solvent extracts, methanol extract has shown the highest activity and its IC50 concentration was found to be 80.8±2.08 μg/ml. The anticancer drug Cis-diammineplatinum (II) dichloride was considered as a standard. Conclusion: In further the bioactive compound should be isolated from the methanol crude extract, which is responsible for the cytotoxic effect

High-purity Isolation for Genotyping Rare Cancer Cells from Blood Using a Microfluidic Chip Cell Sorter

A multistep sorting method for enrichment of rare cells, such as circulating tumor cells, in the blood without cumbersome pretreatments required by most flow cytometry-based methods, which lead to high cost and decreased detection efficiency, was developed. After only hemolysis and cell staining, cancer cells are enriched by repetitive sorting (3×) based on nuclear-positive, cytokeratin-positive, and CD45-negative expression. Experiments using spikes of PC-9 cells showed a mean recovery of 65% and mean purity of 83%, which was retained up to 72 hours after blood draw using preservative tubes. Significant differences in expression level of programmed death-ligand 1 or vimentin were observed between high- and low-expressing cell lines, concurrently with enrichment. Next-generation sequencing analysis of recovered PC-9, A549, and MDA-MB231 cells successfully detected all known mutations. This novel isolation method applicable for preserved samples with sufficient recovery and purity may be substantially beneficial for recovering cells for subsequent molecular analysis.

Synthesis and antiproliferative activity of 6BrCaQ-TPP conjugates for targeting the mitochondrial heat shock protein TRAP1

A series of 6BrCaQ-Cn-TPP conjugates 3a-f and 5 was designed and synthesized as a novel class of TRAP1 inhibitors. Compound 3a displayed an excellent anti-proliferative activity with mean GI50 values at a nanomolar level in a diverse set of human cancer cells (GI50 = 0.008–0.30 μM) including MDA-MB231, HT-29, HCT-116, K562, and PC-3 cancer cell lines. Moreover, the best lead compound 6BrCaQ-C10-TPP induces a significant mitochondrial membrane disturbance combined to a regulation of HSP and partner protein levels as a first evidence that his mechanism of action involves the TRAP-1 mitochondrial Hsp90 machinery.

Trimethyl chitosan coated palladium nanoparticles as a photothermal agent and its in vitro evaluation in 2D and 3D model of breast cancer cells

The potential of palladium has been scantily explored in biomedical applications. In the present study, palladium nanoparticles (PdNPs) were synthesized and were successfully coated with trimethyl-chitosan (TMC) to improve their biocompatibility. Coating with TMC improved the nanoparticle accumulation in MDAMB231 breast cancer cells, compared to nanoparticles coated with native chitosan. The TMC coated palladium nanoparticles (TMC/PdNPs) exhibited good biocompatibility and physiological stability, as compared to the plain(uncoated) PdNPs. TMC coated PdNPs resulted in photothermal therapeutic effect, when irradiated with a near-infrared (NIR) laser having the wavelength of 808-nm. The TMC/PdNPs resulted in good cytotoxic effect upon laser treatment in both, 2D monolayers and 3D spheroids of MDAMB231 cells, the latter mimicking the tumor microenvironment. These results clearly indicated that TMC/PdNPs acted as ideal photothermal agents for anti-cancer therapy in combination with a non-invasive near-infrared laser.

Abstract P127: Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms

Abstract Background: We previously reported that small molecule ONC201 induces mitochondrial structural and functional damage, leading to death in breast cancer cells. Subsequent studies demonstrated that ONC201 and the related analogs TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein for maintenance of mitochondrial protein homeostasis. Recent studies have shown that cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we report that ClpP agonists inhibit breast CSCs by unique mechanisms targeting pathways vital to maintain CSC function. Methods: ONC201 and other ClpP agonists (TR-57, 65), other mitochondria-targeting drugs, such as oligomycin, metformin, CPI-613 were used. MDA-MB231 cell line was used as a primary in vitro model system. CLPP knockout (KO) cells were generated by CRISPR/Cas9 technology. Seahorse XF analyzer was used for cellular respiration analysis. Luminescence-based assays were used for cell viability and metabolism assays. Protein expression was examined by Western blotting. Aldefluor assay and SORE6 (OCT4/SOX2 response element)-reporter gene were used to monitor CSC fraction. Mammosphere formation assay was used to evaluate CSC function in vitro. In vivo limiting dilution analysis was used to evaluate tumor initiation capability of cells injected into mammary fat pad of athymic nude female mice. Results: Seahorse XF analyzer showed that mammospheres are more dependent on OxPhos than glycolysis compared with cells grown in 2D, supporting the hypothesis that breast CSCs rely on OxPhos. ClpP agonists reduced the CSC fraction in both Aldefluor and SORE6 reporter assays. ClpP agonists inhibited mammosphere formation in CLPP WT, but not in CLPP KO cells, demonstrating the on-target effects on CSC function. In in vivo assays, tumor formation was significantly (p&amp;lt;0.001) inhibited in the ClpP agonist-treated group compared with the control group, and the effect was CLPP-dependent. Altogether, these findings support that ClpP agonists inhibit CSC in breast cancers. We found that ClpP agonists downregulate multiple pathways and proteins critical for CSC maintenance including mevalonate pathway, HIF1a, EPAS1, YAP, and Myc. We also observed that other mitochondria targeting drugs such as oligomycin, metformin downregulate these signaling pathways and proteins to some extent. Importantly, however, ClpP agonists showed significantly greater impact in mammosphere formation and cell growth assays, compared with other mitochondria-targeting drugs. Further studies revealed that ClpP agonists uniquely deplete NAD+/NADH and promote reactive oxygen species, both of which are shown as key factors to maintain pluripotency of stem cells. Moreover, ClpP agonists uniquely inhibited enzymes involved with glutamine catabolism and proline biosynthesis, vital to amino acids and nucleotide synthesis. Conclusion: ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells by targeting multiple pathways essential to maintain CSC function. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Donna Voeller, Raj Chari, Binwu Tang, Christina M. Annunziata, Sam Gilbert, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P127.

Yerba Mate Modulates Tumor Cells Functions Involved in Metastasis in Breast Cancer Models.

Breast cancer (BC) is the most frequent cancer in women and tumor metastasis is a major cause of cancer-related deaths. Our aim was to evaluate anti-metastatic properties of yerba mate extract (YMe) in BC models. 4T1, F3II, MCF-7, and MDA-MB231 cell lines were used to perform in vitro assays. The F3II syngeneic mammary carcinoma model in BALB/c mice was used to evaluate tumor progression, BC metastasis and survival. Cells were inoculated subcutaneously into the flank for the heterotopic model and into the mammary fat pad for the orthotopic model. YMe was administered p.o. in a dose of 1.6 g/kg/day. In vitro YMe inhibited cell proliferation and reduced tumor cell adhesion, migration and invasion. These biological effects were cell-line dependent. In vivo YMe reduced tumor metastasis and increased mice survival in both models. Our preclinical results suggest that YMe could modulate tumor progression and metastasis in BC models.