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Lavandula multifida L., commonly known as Egyptian lavender or fern leaf lavender, is widely recognized and used as a medicinal plant throughout Mediterranean countries. While L. multifida has been extensively studied, research has largely centered on its essential oil, with limited data available on the composition and bioactivities of its organic extracts. This study characterized the phytochemical profiles of L. multifida extracts and evaluated their antioxidant capacity, effects on cancer cell viability, and antimicrobial activity using multiple in vitro assays. Phytochemical characterization included the quantification of total phenolic and flavonoid contents and HPLC/ESI-QTOF-MS profiling of methanolic (LM), aqueous (LAQ), and ethyl acetate (LEA) extracts. Antioxidant activity was assessed using four complementary in vitro assays (DPPH, ABTS, FRAP, and TAC). The effects of the extracts on cell viability were assessed using the MTT assay in human triple-negative breast cancer (MDA-MB-231) and colon adenocarcinoma (HCT116) cell lines, while antimicrobial activity was determined using the agar well diffusion and broth microdilution methods. Phytochemical profiling of L. multifida extracts revealed diverse metabolites dominated by fatty acids (mainly oxylipins), together with phenolic acids, flavonoids, terpenes, and other minor constituents. The methanolic extract showed the strongest DPPH scavenging effect (IC₅₀ = 14.19 µg/mL), LAQ showed the highest FRAP value (246.62 µg TE/mg), and LEA demonstrated the greatest ABTS (IC₅₀ = 28.07 µg/mL) and TAC (367.16 µg AAE/mg) activities. LEA also produced the greatest reductions in cell viability, reaching approximately 97% in HCT116 cells and 83% in MDA-MB-231 cells, with corresponding IC₅₀ values of 27.85 ± 2.48 and 63.71 ± 3.05 µg/mL, respectively. LM and LAQ showed the largest bacterial inhibition zones, while LEA, despite weaker diffusion activity, exhibited the greatest potency (MIC = 0.5–2 mg/mL; MBC = 0.5–5 mg/mL) and a unique antifungal effect (MIC = 2 mg/mL; MFC = 4 mg/mL). These findings underscore the potential of L. multifida extracts as a natural reservoir of bioactive compounds with notable antioxidant, anticancer, and antimicrobial activities, suggesting their relevance for future pharmaceutical development. Full article

Background/Objectives: Tarin exhibits immunomodulatory and antiproliferative properties against several tumor cell lines. Nano-encapsulation in liposomes enhances its therapeutic potential by improving protein stability, bioavailability, and sustained release. Previous studies demonstrated that nano-encapsulated tarin induces cell cycle arrest, migration inhibition, apoptosis, and autophagy in triple-negative breast cancer cells; however, the molecular mechanisms underlying these effects remain poorly understood. To investigate the proteomic response elicited by nano-encapsulated tarin, MDA-MB-231 cells were treated for 24 and 48 h. Methods: Intracellular proteins were extracted, digested with trypsin, and analyzed by label-free LC-2D-MS/MS using HDMSE acquisition. Differentially expressed proteins were identified and quantified using the Progenesis QI platform, and then functional classification and pathway enrichment analyses were performed. Results: A total of 2818 proteins were identified, of which 2150 displayed time-dependent modulation following treatment. After 24 h, cells exhibited an adaptive stress response profile characterized by increased DNA repair proteins (CHEK1, CDK12), migration/remodeling factors (LAMA4, CTTN, A2M), and immune/cell cycle regulators (PER2, HLA-B), while antioxidant proteins (SOD1, GPX1) and BRCA1 were reduced, indicating oxidative stress and DNA damage. After 48 h, the proteomic profile shifted toward cell death, with increased PARK7, OPA1, ATL3, and CASP8 expression, disruption of DNA repair and cell cycle regulators (CHEK1, CDK12, MSH6, KIF2C), and decreased migration-related proteins (LAMA4, CTTN, ITGB3, A2M). Conclusions: Nano-encapsulated tarin promotes a time-dependent transition from early adaptive stress responses to apoptosis, autophagy, cell cycle disruption, and loss of migratory capacity. These findings provide novel insights into the molecular mechanisms underlying tarin antitumoral activity and support its potential as a promising therapeutic strategy against triple-negative breast cancer. Full article

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a dose-limiting complication of paclitaxel (PTX) therapy, for which effective treatments are still lacking. This study evaluated the neuroprotective potential of Thymoquinone (TQ), a bioactive derivative of Nigella sativa, in mitigating chronic PTX-induced neurotoxicity without compromising antineoplastic activity. We first performed in vitro experiments using Sprague–Dawley rat embryonic (E15) Dorsal Root Ganglia (DRG) (Envigo Laboratory (Udine, Italy))to assess neurotoxicity through neurite outgrowth evaluation. To investigate the molecular mechanisms underlying TQ’s putative neuroprotective mechanisms, SIRT1 protein expression was additionally evaluated by western blot, while MCF-7 and MDA-MB-231 breast cancer cells were used to monitor cytotoxicity via MTT assay. We then moved to in vivo experiments, in which chronic neuropathy was induced in rats using PTX (10 mg/kg, i.v., weekly for 4 weeks). TQ was co-administered orally (5–10 mg/kg/day). The effects of TQ on peripheral neuropathy were assessed through behavioral testing, neurophysiological assessments, and histological analysis of Intraepidermal Nerve Fibre density (IENF), DRG and peripheral nerves. In vitro, TQ (5 μM) significantly attenuated PTX-induced neurite shortening at 24 h; TQ co-treatment fully prevented PTX-induced SIRT1 downregulation in embryonic DRG neurons and did not compromise PTX cytotoxicity in MCF-7 cells, while significantly potentiating it in MDA-MB-231 triple-negative breast cancer cells. In vivo results demonstrated that PTX-treated animals exhibited mild erythroid myelosuppression at the end of treatment. Regarding efficacy, TQ consistently prevented PTX-induced mechanical allodynia throughout the treatment period, and TQ (10 mg/kg) transiently mitigated IENF depletion at mid-treatment; however, no improvement in neurophysiological parameters or peripheral nerve morphology was observed at either time point. Collectively, these findings suggest that, under conditions of chronic PTX exposure, TQ exerts a predominantly analgesic effect in vivo, without conferring meaningful structural neuroprotection against PTX-induced peripheral nerve degeneration. Full article

Background: Triple-negative breast cancer (TNBC) remains a major cause of cancer mortality due to its aggressive behavior, metabolic adaptability, and high therapeutic resistance. Extracellular vesicles (EVs) within the tumor microenvironment contribute to tumor progression and metastasis by transferring pro-tumorigenic cargo. While conventional Tumor Treating Fields use high-frequency alternating fields to disrupt mitosis, low-energy triple-frequency bioelectromagnetic approaches remain poorly characterized. Methods: We evaluated a device–drug strategy combining triple-frequency low-intensity electromagnetic stimulation (EMS2: 396 Hz, 285 Hz, 528 Hz) with the pleiotropic drug Fingolimod (FTY720). Treatments were tested in MDA-MB-231 and ARM-G breast cancer cells, with Paclitaxel as a positive control. Cell proliferation was assessed by MTS assay, and extracellular vesicles were isolated following individual and combination treatments. Quantitative LC-MS/MS proteomics was used to characterize treatment-induced changes in EVs cargo. Results: EMS2 reduced proliferation in both cell lines and produced morphological changes consistent with altered cell-cycle progression. EMS2 alone triggered adaptive metabolic responses, whereas combination with Fingolimod suppressed these compensatory signatures. EVs proteomics revealed combination-specific alterations associated with mitochondrial stress, ER stress, NF-κB suppression, and autophagy-associated pathways. The combination also reduced levels of metastasis- and stroma-associated proteins, including Mitogen-Activated Protein Kinase 12 (MAPK12) and collagen-associated ECM components (Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type VI Alpha 1 Chain (COL6A1), Collagen Type VI Alpha 3 Chain (COL6A3), and Matrilin 3 (MATN3)) in EVs. Bliss independence analysis identified a subset of metastasis-associated proteins suppressed in EVs beyond the level predicted by an additive model, an exploratory finding that will require further validation with dose–response and functional assays. Conclusions: Combined triple-frequency EMS2 and Fingolimod treatment altered the extracellular vesicle proteome, inducing signatures consistent with mitochondrial and endoplasmic reticulum stress, metabolic disruption, and reduced levels of metastasis-associated and stromal/ECM remodeling proteins, along with reduced proliferation. These findings suggest a coordinated anti-cancer effect of this tunable device–drug strategy, warranting further functional and in vivo validation to confirm therapeutic potential. Full article

Breast cancer remains among the leading causes of incidence and mortality worldwide. Consequently, identifying new treatments and strategies is of critical importance. Evidence indicates that camptothecin and its derivatives may exert anticancer effects in various cancer cell lines, including colon, lung, and ovarian cancers. However, their effects in breast cancer are not yet fully understood. Prior theoretical studies employing docking and molecular dynamics suggest that camptothecin could bind to the HER2 and EGFR receptors, which are overexpressed in breast cancer cells. Investigating interactions between novel molecules with affinity for membrane receptors overexpressed in breast cancer is important for developing personalized therapies and for advancing strategies to selectively target nanomaterials to these cells for diagnostic and therapeutic purposes. This study evaluated the in silico and in vitro effects of camptothecin on the MCF-7 and MDA-MB-231 breast cancer cell lines. Our results show significant inhibition of proliferation and reduced migration at 24, 48, and 72 h in both cell lines. The theoretical analysis indicates high affinity of camptothecin for receptors overexpressed in breast cancer compared with current treatments. Full article

Background/Objectives: Rosmarinic acid (RA) is a naturally occurring polyphenolic compound with promising anticancer activity; however, its potential to enhance the therapeutic efficacy of doxorubicin (DOX) against triple-negative breast cancer (TNBC) has not been comprehensively characterised. This study investigated the pharmacological interaction and associated cellular responses of RA combined with DOX in MDA-MB-231 breast cancer cells, while including HaCaT human keratinocytes as a non-malignant, non-mammary reference model for comparative cytotoxicity assessment. Methods: Cell viability was assessed using the MTT assay, and pharmacological interactions were evaluated using Chou–Talalay combination index (CI) and dose reduction index (DRI) analyses. Intracellular oxidative activity was evaluated using DCFH-DA fluorescence analyses together with N-acetyl-L-cysteine (NAC) modulation experiments. Apoptosis, cell-cycle distribution, mitochondrial membrane potential (JC-1), Caspase-9 immunocytochemistry, nuclear morphology (NucBlue staining), apoptosis- and proliferation-related gene expression (RT-qPCR; BAX, BCL2, CASP3, CASP9, TP53, CDKN1A, MKI67, and PCNA), and bioinformatic pathway analyses were performed to characterise cellular and molecular responses associated with the combined treatment. Results: After 48 h, RA exhibited IC₅₀ values of 188.4 ± 4.2 µM in MDA-MB-231 cells and 146.6 ± 6.8 µM in HaCaT cells, while DOX showed IC₅₀ values of 1.2 ± 0.08 µM and 2.6 ± 0.15 µM, respectively. The Chou–Talalay analysis demonstrated synergistic interactions in MDA-MB-231 cells, with CI values of 0.86, 0.72, and 0.64 at the effect levels of IC₂₅, IC₅₀, and IC₇₅, respectively; the model-derived DOX DRI value at Fa = 0.50 was 1.8. Combination treatment markedly increased intracellular DCFH-DA fluorescence, whereas NAC pretreatment attenuated this signal and partially restored cell viability, supporting a contributory role of intracellular oxidative stress in treatment-associated cytotoxicity. Increased intracellular oxidative activity coincided with mitochondrial membrane depolarisation, increased apoptotic cell death, accumulation of cells in the sub-G₁ phase, enhanced Caspase-9 immunoreactivity, and pronounced apoptotic nuclear alterations. RT-qPCR analysis demonstrated significant upregulation of BAX, CASP3, CASP9, and TP53, together with downregulation of BCL2, MKI67, and PCNA, resulting in a marked reduction in the BCL2/BAX mRNA expression ratio. A modest but non-significant increase in CDKN1A expression was also observed. Bioinformatic analyses further identified predicted associations with mitochondrial apoptosis and p53-associated signalling pathways. Conclusions: The RA + DOX combination showed synergistic cytotoxic activity in MDA-MB-231 cells, accompanied by increased intracellular oxidative activity, mitochondrial membrane depolarisation, apoptosis, cell-cycle perturbation, and changes in proliferation-related gene expression. These findings indicate associated cellular responses but do not establish a ROS-dependent mitochondrial apoptotic mechanism. The findings provide an exploratory in vitro basis for further investigation of the RA + DOX combination in additional TNBC models, non-malignant mammary epithelial cells, and appropriate in vivo systems. Full article

Background: Building on earlier work with methoxy-, dimethoxy-, bromo-, and nitro-substituted salicylaldehyde benzoylhydrazones, we synthesized and evaluated new 4-chloro- and 5-chloro derivatives bearing an additional chlorine substituent on the hydrazide-derived phenyl ring to further explore the impact of halogenation on cytotoxic activity. Methods: Chloro-hydrazones were obtained through a one-step condensation of 4- or 5-chlorosalicylaldehyde with benzhydrazide or 2-, 3-, and 4-chlorobenzhydrazides. Their physicochemical, pharmacokinetic, ADME, lead-likeness, and drug-likeness profiles were evaluated in silico using SwissADME, ACD/Labs v9.10, and MDL QSAR v2.2.0.0.446. The synthesized compounds were structurally characterized by IR, ¹H NMR, ¹³C NMR, and HR ESI–MS, and their cytotoxic activity was subsequently assessed by the MTT assay in selected cancer cell lines. Molecular docking was performed against ABL1 tyrosine kinase (ABL1 TK), a potential molecular target relevant to two of the investigated leukemia cell lines, using GOLD v.5.2.2 (CCDC Ltd., Cambridge, UK). Results: The chloro-substituted salicylaldehyde benzoylhydrazones exhibited pronounced, cell-type-dependent cytotoxicity, with leukemia cells being substantially more sensitive than breast carcinoma cells. Most derivatives showed low- to sub-micromolar IC₅₀ values against SKW-3 human T-cell prolymphocytic leukemia, K-562 human chronic myeloid (myelogenous) leukemia, and BV-173 human BCR::ABL1-positive leukemia cells, with K3 and K4 exhibiting the highest activity in SKW-3 cells (IC₅₀ = 0.5 ± 0.1 µM). K5 showed particularly strong activity against K-562 and BV-173 cells (IC₅₀ = 0.7 ± 0.1 and 0.9 ± 0.1 µM, respectively), compared with 26.9 ± 2.4 and 21.5 ± 3.3 µM for imatinib. HL-60 human acute promyelocytic leukemia cells showed intermediate sensitivity (IC₅₀ = 1.3–13.9 µM), whereas the activity against MCF-7 estrogen receptor-positive and MDA-MB-231 triple-negative breast carcinoma cells was more variable (1.5–32.1 and 4.1–62.5 µM, respectively). The derivatives showed high selectivity toward malignant cells relative to non-malignant CCL-1 non-malignant mouse fibroblasts, with lower-bound SI values frequently exceeding 50 and reaching >200 in SKW-3 cells. Conclusions: The chloro-substituted salicylaldehyde benzoylhydrazones exhibited pronounced and selective cytotoxicity, particularly toward leukemia cell lines, identifying this scaffold as a promising starting point for further anticancer drug-discovery studies. Full article

Although cryoablation of tumor tissue is known to induce systemic antitumor immune responses, the underlying mechanisms remain poorly understood, and therapeutic efficacy is often unpredictable. During cryoablation, the cryoprobe creates a temperature gradient, resulting in a central zone of lethal tissue destruction and a periphery characterized by sub-lethal injury. In this peripheral zone, cells undergo programmed cell death triggered by thermal shock and ischemia. A widely cited, yet debated, hypothesis suggests that these apoptotic events in the tumor periphery may suppress the antitumor immune response. The purpose of this study was to experimentally test this hypothesis. We utilized the murine breast cancer cell line (4T1-Luc) and a panel of human cell lines (MCF-7, BT-20, BT-474, MDA-MB-231, MDA-MB-453, HBL-100). Following validation via flow cytometry using Annexin V-AF488, Rhodamine 123, TMRE, PI, and 7-AAD, we established −7 °C and −80 °C as temperature conditions that predominantly induce apoptosis and necrosis, respectively. These conditions were used to prepare cryo-treated 4T1-Luc cells for the vaccination of syngeneic mice. Ten days post-immunization, the efficacy of the treatment was evaluated in subcutaneous solid tumor and lung metastasis models by monitoring tumor growth, quantifying tumor-specific antibodies, performing histological analysis of tumor and lung tissues, and quantifying lung metastases via PCR and luciferase assays. Contrary to the initial hypothesis, our findings demonstrate that tumor cells undergoing sub-lethal cryoablation do not acquire immunosuppressive characteristics. Instead, they promote systemic antitumor immunity. Although antitumor immunity induced by apoptotic and necrotic tumor cells was comparable in a subcutaneous tumor model, vaccination with necrotic tumor cells proved more effective than vaccination with apoptotic cells in controlling lung metastasis formation. Full article

Background/Objectives: Breast cancer exhibits substantial molecular heterogeneity, resulting in diverse therapeutic vulnerabilities and limiting the efficacy of single-agent therapies. Although multi-target strategies may offer improved therapeutic benefit, the systematic identification of synergistic higher-order target combinations remains challenging. This study aimed to identify and validate effective higher-order target combinations for heterogeneous breast cancer. Methods: DeepMDS, a previously developed deep learning-based multi-compound synergy prediction model, was used to prioritize candidate target combinations for breast cancer. Exhaustive two-target and three-target combinations were ranked in the gene-expression contexts of ER-positive luminal-like MCF-7 and triple-negative MDA-MB-231 breast cancer cells. The top-ranked target combinations were evaluated using single, pairwise, and triple small interfering RNA (siRNA) perturbations. Representative inhibitors were subsequently assessed in fixed-ratio combinations in MCF-7, MDA-MB-231, and 4T1 cells and in a 4T1 syngeneic mouse experiment. Results: BIRC5-NAMPT-TOP1 ranked first in both cell lines. Triple siRNA co-transfection targeting BIRC5, NAMPT, and TOP1 produced the strongest antiproliferative effects, with inhibition rates of 62.94% in MCF-7 cells and 55.62% in MDA-MB-231 cells. The corresponding inhibitors, LQZ-7I, FK866, and topotecan, exhibited synergistic antiproliferative activity at multiple molar ratios, with combination index values below 1. The optimized 2.5:10:1 molar ratio showed strong synergy in MCF-7, MDA-MB-231, and 4T1 cells, with combination index values of 0.26, 0.19, and 0.15, respectively. In tumor-bearing mice model, the triple-inhibitor regimen achieved a tumor inhibition rate of 62.05%. Topotecan-containing groups showed hematological alterations, whereas no statistically detectable elevations were observed in the measured terminal serum hepatic or renal biomarkers. Conclusions: The BIRC5-NAMPT-TOP1 combination showed reproducible phenotypic activity in the tested genetic and pharmacological models. These findings support the use of DeepMDS as a hypothesis-generation tool for higher-order target prioritization. Full article

Background/Objectives: Postbiotics derived from beneficial bacteria have emerged as promising bioactive ingredients owing to their enhanced stability, safety, and functional versatility relative to live microorganisms. Methods: The present study was structured in two complementary stages: (i) biological characterization of liquid microfiltrates obtained from Lactiplantibacillus plantarum and Bacillus mojavensis fermented in aguamiel, and (ii) technological stabilization of these postbiotics through microencapsulation by spray drying, with a view to their potential incorporation into food matrices. Results&Conclusions: In the first stage, biological activity was assessed in vitro using MDA-MB-231 human breast cancer cells and in vivo in C57BL/6J mice, in order to evaluate safety and gastrointestinal effects. Both postbiotics significantly reduced cell viability relative to controls, consistent with antiproliferative activity. In vivo, no macroscopic intestinal damage was observed, supporting their safety profile. Histomorphometric analysis revealed preserved intestinal architecture, with elongation of villi and crypts and increased mucin 2 production, indicating strengthened mucosal barrier integrity; these effects were more pronounced in the Bacillus mojavensis-treated group. In the second stage, the spray-dried powders exhibited adequate physicochemical stability, supporting their potential application as functional food ingredients. The spray-dried powders exhibited adequate physicochemical stability, supporting their potential application as functional food ingredients. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) exhibits substantial metabolic heterogeneity and plasticity, contributing to variable therapeutic responses. We investigated whether optical redox imaging (ORI) could characterize metabolic phenotypes, monitor responses to metabolic perturbation, and relate these responses to functional outcomes in TNBC cells. Methods: Four TNBC cell lines were treated with the lactate dehydrogenase A inhibitor FX11 or the glutaminase inhibitor CB-839, alone or in combination with paclitaxel. Intensity-based label-free ORI was performed and followed by imaging of fluorescent probes to assess mitochondrial membrane potential (MMP), re-active oxygen species (ROS), and cell number in the same dishes. Seahorse assays were used to evaluate mitochondrial respiration and glycolytic flux. Results: TNBC cell lines exhibited distinct basal redox phenotypes and differential responses to acute glycolytic and glutaminolytic perturbation. HCC1806 cells showed the strongest acute ORI responses to both FX11 and CB-839. Acute FX11 treatment induced a rapid reductive shift accompanied by ROS accumulation and loss of MMP, whereas CB-839 produced a more modest early reductive response without detectable ROS accumulation or MMP loss. Prolonged treatment revealed distinct temporal redox trajectories in HCC1806 cells: FX11-treated cells evolved from an acute reductive response toward a more oxidized state, while CB-839-treated cells transitioned from an early reductive shift to a sustained oxidized redox state accompanied by marked reductions in OCR and ECAR. Functionally, in two representative models (HCC1806 and MDA-MB-231), CB-839 reduced cell numbers and enhanced the anti-proliferative effect of paclitaxel, whereas FX11 had no significant effect on cell number despite inducing pronounced acute redox perturbations. Conclusions: Integrating ORI with metabolic flux assays and imaging-based functional measurements enables characterization of multiple dimensions of metabolic behavior, including basal phenotype, pathway-specific responsiveness, temporal redox responses, and treatment-associated outcomes. These findings support intensity-based wide-field ORI as a practical and accessible tool for probing metabolic heterogeneity and characterizing context-dependent metabolic responses in TNBC cells. Full article

Background/Objectives: The health-protective properties of olive oil phenolics, including their potential chemopreventive and anticancer effects, have attracted considerable scientific interest. However, their in vivo efficacy and mechanisms of action remain insufficiently understood. Recent advances in extraction and purification technologies have enabled large-scale production of highly purified olive oil phenols and phenolic-rich extracts, facilitating translational research. Methods: Herein, the antitumor efficacy of isolated olive oil phenols and phenolic-rich formulations was investigated in an MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model. Results: Intraperitoneal administration of oleocanthal (OLC), oleuropein aglycone (OleA) or their combination reduced endpoint tumor burden, with OLC exhibiting the most pronounced effect. Oral administration of total olive oil phenolics (OOPs) achieved comparable efficacy. Pre-treatment initiated before tumor cell implantation conferred the greatest protection, consistent with a prophylactic mode of action. In contrast, delayed intervention displayed diminished or no antitumor benefit. Phenolic-rich extra virgin olive oil likewise showed an inhibition trend in tumor progression. Mechanistically, OOPs attenuated plasma protein oxidation, modulated proteasome mediated proteolysis, and reduced γH2AX levels in vivo. Furthermore, OOPs negatively affected the MDA-MB-231 cell migration in a concentration-dependent manner in vitro. Conclusions: Collectively, these findings are consistent with antitumor activities of olive oil phenolics via multiple mechanisms and support their further investigation as prophylactic agents in TNBC and as nutraceuticals. Full article

Breast cancer cells exhibit a reversed pH gradient and metabolic plasticity that promote proliferation, invasion, and resistance to therapy. Natural products such as chlorogenic acid (CGA) and cinnamaldehyde (CA) have shown emerging anticancer potential. However, their effects on intracellular pH and metabolic transport systems remain undefined. Therefore, the aim of this study was to characterize these parameters in breast cancer and non-tumorigenic breast cells. This study evaluated the physiochemical properties of CGA and CA using LC–MS, under pH conditions (pH 1.2, 7.4, and 9.0) mimicking the gastrointestinal track (GIT). Additionally, LC–MS-based human liver microsome (HLM) assays with NADPH were used to evaluate susceptibility to CYP-mediated metabolism to evaluate first-pass metabolic stability. Intracellular uptake kinetics were quantified at multiple time points using LC–MS. Following CGA:CA treatment, intracellular pH (pH_i) was measured in cancerous MDA-MB-231 and non-tumorigenic MCF-10A breast cell lines using SNARF-1 targeted ratio-metric fluorescence approach. Expression of OATP1B1, GLUT1, and MCT1 were analyzed by Western and immunofluorescence respectively, to assess potential cellular uptake of CGA:CA through OATP1B1 and their effects on glucose uptake and lactate and proton transport. Physiochemical results demonstrated that the compounds ranged from fully stable (pH 1.2 and 7.4) to completely unstable (pH 9.0). HLM incubation indicated no CYP-mediated hepatic metabolism. Treatment results showed that there was rapid intracellular uptake of CGA and CA in cancer cells and that CGA:CA lowered pH_i in both MDA-MB-231 and MCF-7 cells, while pH_i remained mostly unchanged in MCF-10A cells. Protein analysis revealed that CGA:CA treatment downregulated GLUT1 and MCT1 expression in cancer cells, suggesting impaired glycolytic activity and lactate shuttling. OATP1B1 expression was significantly suppressed in cancer cells, suggesting feedback inhibition of the solute carrier protein. Collectively, these findings indicate that CGA and CA exhibit favorable biochemical stability and disrupt intracellular pH regulation and metabolic transporter expression in breast cancer cells. Importantly, normal cells are not significantly affected. Thus, CGA:CA demonstrates therapeutic potential for breast cancer through pH_i and metabolic modulation. Full article

Cancer, the second leading cause of death worldwide, is a significant global challenge, and widespread, accessible, and early diagnostics are recognized as the most cost-effective strategies for reducing cancer burdens. Point-of-care (POC) systems offer an attractive alternative by enabling rapid and cost-effective diagnoses. We introduce a novel POC strategy for cancer biomarker identification based on monitoring the isothermal amplification of relevant cancer markers using a portable Arduino-based loop-mediated isothermal amplification (LAMP) system. The trajectory of the LAMP reaction during the first 3 min of the reaction is used as an indicator of the rate of amplification (defined as the m_P3 value). We obtained sets of m_P3 values that showed statistically significant differences in the genetic expression of four genes (ESR 1, PGR, Her2, and Ki67) within and between tissue spheroids derived from the MCF7, MDA-MB-231, Du145, and BJ fibroblast cell lines. We then used principal component analysis and clustering techniques to demonstrate that the m_P3 value sets derived from the expression of the four selected genes are sufficient to distinguish tissue spheroids derived from four different commercial cell lines. Further qPCR and immunostaining assays confirmed the quantitative LAMP (qLAMP) experimental trends. The immunostaining results were consistent with previous literature reports and with our qLAMP and qPCR results. We present a proof-of-concept demonstration of the use of a LAMP-based POC platform for the identification or discrimination of cancer tissues. Our strategy can be extended to other diseases associated with altered gene expression in body tissues or fluids. Full article

Background/Objectives: Triple negative breast cancer remains the most difficult breast cancer to treat as there are no specific targeted therapies. The tumor stroma is recognized as essential for the development of solid tumors. Cancer associated myofibroblasts are known to play a major role in the construction of a tumor microenvironment conducive to tumor progression. Therefore, we established a co-culture system to identify potential molecular targets controlling myofibroblast conversion. Methods: Co-cultures of MDA-MB-468 triple negative breast cancer cells and skin fibroblasts were either mixed prior to seeding or seeded individually on opposite sides of a culture dish and incubated in complete medium. Inhibitors were preincubated with cells for 1 h. Inflammatory cytokines were measured by ELISA. Proteins were analyzed using the Wes-Protein Simple System. Immunostaining was visualized using a Hamamatsu Photonics Nanozoomer. Statistical analysis was done by student’s t-test and one-way ANOVA. Results: The data indicate that co-culturing the cells promotes myofibroblast differentiation and cytokine release and this requires direct contact between the two cell types. Both myofibroblast conversion and cytokine release were prevented by inhibitors of TLR5, TGF-β and EDA-fibronectin. Conclusions: Data suggest that the TLR5 receptor on the MDA-MB-468 cells binds to EDA-fibronectin expressed by fibroblasts thus inducing inflammatory cytokine expression. Expression of EDA fibronectin is regulated through TGF-β whose synthesis is induced by TLR5 signaling in the tumor cells. The data are consistent with a model in which TLR5-mediated crosstalk between the cancer cells and the stromal fibroblasts creates a tissue microenvironment conducive to tumor growth and metastasis. Full article