Caspase-9 suppresses metastatic behavior of MDA-MB-231 cells in an adaptive organoid model.

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Multiparametric MRI-based radiomic models for early prediction of response to neoadjuvant systemic therapy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is often treated with neoadjuvant systemic therapy (NAST). We investigated if radiomic models based on multiparametric Magnetic Resonance Imaging (MRI) obtained early during NAST predict pathologic complete response (pCR). We included 163 patients with stage I-III TNBC with multiparametric MRI at baseline and after 2 (C2) and 4 cycles of NAST. Seventy-eight patients (48%) had pCR, and 85 (52%) had non-pCR. Thirty-six multivariate models combining radiomic features from dynamic contrast-enhanced MRI and diffusion-weighted imaging had an area under the receiver operating characteristics curve (AUC) > 0.7. The top-performing model combined 35 radiomic features of relative difference between C2 and baseline; had an AUC = 0.905 in the training and AUC = 0.802 in the testing set. There was high inter-reader agreement and very similar AUC values of the pCR prediction models for the 2 readers. Our data supports multiparametric MRI-based radiomic models for early prediction of NAST response in TNBC.

Inhibition of Cancer Stem-like Cells by Curcumin and Other Polyphenol Derivatives in MDA-MB-231 TNBC Cells.

Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers and is highly aggressive. Despite an initial positive response to chemotherapy, most patients experience rapid disease progression leading to relapse and metastasis. This is attributed to the presence of breast cancer stem cells (BCSCs) within the tumor, which are characterized by self-renewal, pluripotency, and resistance mechanisms. Targeting BCSCs has become critical as conventional therapies fail to eradicate them due to a lack of specific targets. Curcumin, a polyphenol derived from turmeric (Curcuma longa), exhibits anticancer effects against breast cancer cells and BCSCs. The use of curcumin derivatives has been suggested as an approach to overcome the bioavailability and solubility problems of curcumin in humans, thereby increasing its anticancer effects. The aim of this study was to evaluate the cellular and molecular effects of six synthetic compounds derived from the natural polyphenol epigallocatechin gallate (EGCG) (TL1, TL2) and curcumin derivatives (TL3, TL4, TL5, and TL6) on a TNBC mesenchymal stem-like cell line. The activity of the compounds against BCSCs was also determined by a mammosphere inhibition assay and studying different BCSC markers by Western blotting. Finally, a drug combination assay was performed with the most promising compounds to evaluate their potential synergistic effects with the chemotherapeutic agents doxorubicin, cisplatin, and paclitaxel. The results showed that compounds exhibited specific cytotoxicity against the TNBC cell line and BCSCs. Interestingly, the combination of the curcumin derivative TL3 with doxorubicin and cisplatin displayed a synergistic effect in TNBC cells.

In silico fragment-based discovery of CIB1-directed anti-tumor agents by FRASE-bot.

Chemical probes are an indispensable tool for translating biological discoveries into new therapies, though are increasingly difficult to identify since novel therapeutic targets are often hard-to-drug proteins. We introduce FRASE-based hit-finding robot (FRASE-bot), to expedite drug discovery for unconventional therapeutic targets. FRASE-bot mines available 3D structures of ligand-protein complexes to create a database of FRAgments in Structural Environments (FRASE). The FRASE database can be screened to identify structural environments similar to those in the target protein and seed the target structure with relevant ligand fragments. A neural network model is used to retain fragments with the highest likelihood of being native binders. The seeded fragments then inform ultra-large-scale virtual screening of commercially available compounds. We apply FRASE-bot to identify ligands for Calcium and Integrin Binding protein 1 (CIB1), a promising drug target implicated in triple negative breast cancer. FRASE-based virtual screening identifies a small-molecule CIB1 ligand (with binding confirmed in a TR-FRET assay) showing specific cell-killing activity in CIB1-dependent cancer cells, but not in CIB1-depletion-insensitive cells.

The therapeutic efficacy of 5-ALA based photodynamic therapy and chemotherapy combination in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is one of the subtypes of breast cancer characterized by a heterogeneous and aggressive nature. Photodynamic therapy (PDT) has drawn significant attention in cancer treatment. However, solubility of photosensitizer, penetration problems into a target tissue and insufficient oxygen concentration limit the effectiveness of PDT. To overcome these limitations and to reduce the side effects of chemotherapy, combination treatment modalities play an essential role in cancer treatment. In this study, we aimed to investigate the combination efficacy of cisplatin-based chemotherapy and 5-Aminolevulinic acid (5-ALA)/PDT in TNBC cells and healthy breast cells in vitro. To determine the effect of the combination effects of cisplatin and 5-ALA/PDT on TNBC cells, two treatment protocols (simultaneous and sequential combination therapy) were evaluated compared with cisplatin and 5-ALA/PDT monotherapy and WST-1, Annexin V assay, acridine orange (AO) and mitochondrial staining were performed. Our findings showed that MDA-MB-231 TNBC cell viability was significantly decreased following simultaneous combination treatment compared to cisplatin and 5-ALA/PDT monotherapy. Additionally, simultaneous combination treatment was more effective than sequential combination treatment. The simultaneous combination treatment of 2.5 µM cisplatin and 5-ALA/PDT at 6 J/cm2 and 9 J/cm2 induced 46.78% and 53.6% total apoptotic death, respectively in TNBC cells compared with monotherapies (cisplatin (37.88%) and 5-ALA/PDT (6 J/cm2: 31.48% and 9 J/cm2: 37.78%). Additionally, cisplatin and 5-ALA/PDT combination treatment resulted in nuclear fragmentation and mitochondrial damage due to apoptosis. Our results suggest that cisplatin and 5-ALA/PDT simultaneous combination therapy could be a promising new alternative strategy for treating TNBC. However, further studies are required to assess the underlying molecular mechanisms of cisplatin and 5-ALA/PDT combination treatment at the molecular level.

Tumor necrosis factor receptor 2 promotes endothelial cell-mediated suppression of CD8+ T cells through tuning glycolysis in chemoresistance of breast cancer.

BACKGROUND: T cells play a pivotal role in chemotherapy-triggered anti-tumor effects. Emerging evidence underscores the link between impaired anti-tumor immune responses and resistance to paclitaxel therapy in triple-negative breast cancer (TNBC). Tumor-related endothelial cells (ECs) have potential immunoregulatory activity. However, how ECs regulate T cell activity during TNBC chemotherapy remains poorly understood. METHODS: Single-cell analysis of ECs in patients with TNBC receiving paclitaxel therapy was performed using an accessible single-cell RNA sequencing (scRNA-seq) dataset to identify key EC subtypes and their immune characteristics. An integrated analysis of a tumor-bearing mouse model, immunofluorescence, and a spatial transcriptome dataset revealed the spatial relationship between ECs, especially Tumor necrosis factor receptor (TNFR) 2+ ECs, and CD8+ T cells. RNA sequencing, CD8+ T cell proliferation assays, flow cytometry, and bioinformatic analyses were performed to explore the immunosuppressive function of TNFR2 in ECs. The downstream metabolic mechanism of TNFR2 was further investigated using RNA sequencing, cellular glycolysis assays, and western blotting. RESULTS: In this study, we identified an immunoregulatory EC subtype, characterized by enhanced TNFR2 expression in non-responders. By a mouse model of TNBC, we revealed a dynamic reduction in the proportion of the CD8+ T cell-contacting tumor vessels that could co-localize spatially with CD8+ T cells during chemotherapy and an increased expression of TNFR2 by ECs. TNFR2 suppresses glycolytic activity in ECs by activating NF-κB signaling in vitro. Tuning endothelial glycolysis enhances programmed death-ligand (PD-L) 1-dependent inhibitory capacity, thereby inducing CD8+ T cell suppression. In addition, TNFR2+ ECs showed a greater spatial affinity for exhausted CD8+ T cells than for non-exhausted CD8+ T cells. TNFR2 blockade restores impaired anti-tumor immunity in vivo, leading to the loss of PD-L1 expression by ECs and enhancement of CD8+ T cell infiltration into the tumors. CONCLUSIONS: These findings reveal the suppression of CD8+ T cells by ECs in chemoresistance and indicate the critical role of TNFR2 in driving the immunosuppressive capacity of ECs via tuning glycolysis. Targeting endothelial TNFR2 may serve as a potent strategy for treating TNBC with paclitaxel.

Identification of New Chemoresistance-Associated Genes in Triple-Negative Breast Cancer by Single-Cell Transcriptomic Analysis.

Triple-negative breast cancer (TNBC) is a particularly aggressive mammary neoplasia with a high fatality rate, mainly because of the development of resistance to administered chemotherapy, the standard treatment for this disease. In this study, we employ both bulk RNA-sequencing and single-cell RNA-sequencing (scRNA-seq) to investigate the transcriptional landscape of TNBC cells cultured in two-dimensional monolayers or three-dimensional spheroids, before and after developing resistance to the chemotherapeutic agents paclitaxel and doxorubicin. Our findings reveal significant transcriptional heterogeneity within the TNBC cell populations, with the scRNA-seq identifying rare subsets of cells that express resistance-associated genes not detected by the bulk RNA-seq. Furthermore, we observe a partial shift towards a highly mesenchymal phenotype in chemoresistant cells, suggesting the epithelial-to-mesenchymal transition (EMT) as a prevalent mechanism of resistance in subgroups of these cells. These insights highlight potential therapeutic targets, such as the PDGF signaling pathway mediating EMT, which could be exploited in this setting. Our study underscores the importance of single-cell approaches in understanding tumor heterogeneity and developing more effective, personalized treatment strategies to overcome chemoresistance in TNBC.

Functionalized solid lipid nanoparticles combining docetaxel and erlotinib synergize the anticancer efficacy against triple-negative breast cancer.

The goal of the study was to fabricate folic acid functionalized docetaxel (DOC)/erlotinib (ERL)-loaded solid lipid nanoparticles (SLNs) to synergistically increase the anticancer activity against triple-negative breast cancer. DOC/ERL-SLNs were prepared by the high shear homogenization - ultrasound dispersion method (0.1 % w/v for DOC, and 0.3 %w/v for ERL) and optimized using Plackett Burman Design (PBD) followed by Box Behnken Design (BBD). The optimized SLNs demonstrated particle size < 200 nm, PDI < 0.35, and negative zeta potential with entrapment and loading efficiency of ∼80 and ∼4 %, respectively. The SLNs and folic acid functionalized SLNs (FA-SLNs) showed sustained release for both drugs, followed by Higuchi and Korsemeyer-Peppas drug release models, respectively. Further, the in vitro pH-stat lipolysis model demonstrated an approximately 3-fold increase in the bioaccessibility of drugs from SLNs compared to suspension. The TEM images revealed the spherical morphology of the SLNs. DOC/ERL loaded SLNs showed dose- and time-dependent cytotoxicity and exhibited a synergism at a molar ratio of 1:3 in TNBC with a combination index of 0.35 and 0.37, respectively. FA-DOC/ERL-SLNs showed enhanced anticancer activity as evidenced by MMP and ROS assay and further inhibited the colony-forming ability and the migration capacity of TNBC cells. Conclusively, the study has shown that SLNs are encouraging systems to improve the pharmaceutical attributes of poorly bioavailable drugs.

Single-cell transcriptome sequencing reveals SPP1-CD44-mediated macrophage-tumor cell interactions drive chemoresistance in TNBC.

Triple-negative breast cancer (TNBC) is often considered one of the most aggressive subtypes of breast cancer, characterized by a high recurrence rate and low overall survival (OS). It is notorious for posing challenges related to drug resistance. While there has been progress in TNBC research, the mechanisms underlying chemotherapy resistance in TNBC remain largely elusive. We collect single-cell RNA sequencing (scRNA-seq) data from five TNBC patients susceptible to chemotherapy and five resistant cases. Comprehensive analyses involving copy number variation (CNV), pseudotime trajectory, cell-cell interactions, pseudospace analysis, as well as transcription factor and functional enrichment are conducted specifically on macrophages and malignant cells. Furthermore, we performed validation experiments on clinical samples using multiplex immunofluorescence. We identified a subset of SPP1+ macrophages that secrete SPP1 signals interacting with CD44 on malignant cell surfaces, potentially activating the PDE3B pathway within malignant cells via the integrin pathway, leading to chemotherapy resistance. The abnormally enhanced SPP1 signal between macrophages and malignant cells may serve as a factor promoting chemotherapy resistance in TNBC patients. Therefore, SPP1+ macrophages could potentially serve as a therapeutic target to reduce chemotherapy resistance.

Novel insights into paclitaxel's role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment.

BACKGROUND: Triple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed cell death protein-1 (PD-1) blockade in treating TNBC, although the exact mechanisms behind the improved results are unclear. METHODS: We employed an integrated approach, analyzing spatial transcriptomics and single-cell RNA sequencing data from TNBC patients to understand why the combination of PTX and PD-1 blockade showed better response in TNBC patients. We focused on toll-like receptor 4 (TLR4), a receptor of PTX, and its role in modulating the cross-presentation signaling pathways in tumor-associated macrophages (TAMs) within the tumor microenvironment. Leveraging insights obtained from patient-derived data, we conducted in vitro experiments using immunosuppressive bone marrow-derived macrophages (iBMDMs) to validate if PTX could augment the cross-presentation and phagocytosis activities. Subsequently, we extended our study to an in vivo murine model of TNBC to ascertain the effects of PTX on the cross-presentation capabilities of TAMs and its downstream impact on CD8+ T cell-mediated immune responses. RESULTS: Data analysis from TNBC patients revealed that the activation of TLR4 and cross-presentation signaling pathways are crucial for the antitumor efficacy of PTX. In vitro studies showed that PTX treatment enhances the cross-presentation ability of iBMDMs. In vivo experiments demonstrated that PTX activates TLR4-dependent cross-presentation in TAMs, improving CD8+ T cell-mediated antitumor responses. The efficacy of PTX in promoting antitumor immunity was elicited when combined with PD-1 blockade, suggesting a complementary interaction. CONCLUSIONS: This study reveals how PTX boosts the effectiveness of PD-1 inhibitors in treating TNBC. We found that PTX activates TLR4 signaling in TAMs. This activation enhances their ability to present antigens, thereby boosting CD8+ T cell antitumor responses. These findings not only shed light on PTX's immunomodulatory role in TNBC but also underscore the potential of targeting TAMs' antigen presentation capabilities in immunotherapy approaches.

Exploring chromone-2-carboxamide derivatives for triple-negative breast cancer targeting EGFR, FGFR3, and VEGF pathways: Design, synthesis, and preclinical insights.

Chromone-based compounds have established cytotoxic, antiproliferative, antimetastatic, and antiangiogenic effects on various cancer cell types via modulating different molecular targets. Herein, 17 novel chromone-2-carboxamide derivatives were synthesized and evaluated for their in vitro anticancer activity against 15 human cancer cell lines. Among the tested cell lines, MDA-MB-231, the triple-negative breast cancer cell line, was found to be the most sensitive, where the N-(2-furylmethylene) (15) and the α-methylated N-benzyl (17) derivatives demonstrated the highest growth inhibition with GI50 values of 14.8 and 17.1 µM, respectively. In vitro mechanistic studies confirmed the significant roles of compounds 15 and 17 in the induction of apoptosis and suppression of EGFR, FGFR3, and VEGF protein levels in MDA-MB-231 cancer cells. Moreover, compound 15 exerted cell cycle arrest at both the G0-G1 and G2-M phases. The in vivo efficacy of compound 15 as an antitumor agent was further investigated in female mice bearing Solid Ehrlich Carcinoma. Notably, administration of compound 15 resulted in a marked decrease in both tumor weight and volume, accompanied by improvements in biochemical, hematological, histological, and immunohistochemical parameters that verified the repression of both angiogenesis and inflammation as additional Anticancer mechanisms. Moreover, the binding interactions of compounds 15 and 17 within the binding sites of all three target receptors (EGFR, FGFR3, and VEGF) were clearly illustrated using molecular docking.

Advancing targeted combination chemotherapy in triple negative breast cancer: nucleolin aptamer-mediated controlled drug release.

BACKGROUND: Triple-negative breast cancer (TNBC) is a recurrent, heterogeneous, and invasive form of breast cancer. The treatment of TNBC patients with paclitaxel and fluorouracil in a sequential manner has shown promising outcomes. However, it is challenging to deliver these chemotherapeutic agents sequentially to TNBC tumors. We aim to explore a precision therapy strategy for TNBC through the sequential delivery of paclitaxel and fluorouracil. METHODS: We developed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The binding affinity to the target protein was validated using Enzyme-linked oligonucleotide assays and Surface plasmon resonance assays. The targeting and internalization abilities into tumors were confirmed using Flow cytometry assays and Confocal microscopy assays. The inhibitory effects on TNBC progression were evaluated by pharmacological studies in vitro and in vivo. RESULTS: Various redox-responsive aptamer-paclitaxel conjugates were synthesized. Among them, AS1411-paclitaxel conjugate with a thioether linker (ASP) exhibited high anti-proliferation ability against TNBC cells, and its targeting ability was further improved through fluorouracil modification. The fluorouracil modified AS1411-paclitaxel conjugate with a thioether linker (FASP) exhibited effective targeting of TNBC cells and significantly improved the inhibitory effects on TNBC progression in vitro and in vivo. CONCLUSIONS: This study successfully developed fluorouracil-modified AS1411-paclitaxel conjugates with a thioether linker for targeted combination chemotherapy in TNBC. These conjugates demonstrated efficient recognition of TNBC cells, enabling targeted delivery and controlled release of paclitaxel and fluorouracil. This approach resulted in synergistic antitumor effects and reduced toxicity in vivo. However, challenges related to stability, immunogenicity, and scalability need to be further investigated for future translational applications.

PKMYT1 knockdown inhibits cholesterol biosynthesis and promotes the drug sensitivity of triple-negative breast cancer cells to atorvastatin.

Triple negative breast cancer (TNBC) as the most aggressive molecular subtype of breast cancer is characterized by high cancer cell proliferation and poor patient prognosis. Abnormal lipid metabolism contributes to the malignant process of cancers. Study observed significantly enhanced cholesterol biosynthesis in TNBC. However, the mechanisms underlying the abnormal increase of cholesterol biosynthesis in TNBC are still unclear. Hence, we identified a member of the serine/threonine protein kinase family PKMYT1 as a key driver of cholesterol synthesis in TNBC cells. Aberrantly high-expressed PKMYT1 in TNBC was indicative of unfavorable prognostic outcomes. In addition, PKMYT1 promoted sterol regulatory element-binding protein 2 (SREBP2)-mediated expression of enzymes related to cholesterol biosynthesis through activating the TNF/ TNF receptor-associated factor 1 (TRAF1)/AKT pathway. Notably, downregulation of PKMYT1 significantly inhibited the feedback upregulation of statin-mediated cholesterol biosynthesis, whereas knockdown of PKMYT1 promoted the drug sensitivity of atorvastatin in TNBC cells. Overall, our study revealed a novel function of PKMYT1 in TNBC cholesterol biosynthesis, providing a new target for targeting tumor metabolic reprogramming in the cancer.

A pH-triggered N-oxide polyzwitterionic nano-drug loaded system for the anti-tumor immunity activation research.

Triple negative breast cancer (TNBC) has the characteristics of low immune cell infiltration, high expression of tumor programmed death ligand 1 (PD-L1), and abundant cancer stem cells. Systemic toxicity of traditional chemotherapy drugs due to poor drug selectivity, and chemotherapy failure due to tumor drug resistance and other problems, so it is particularly important to find new cancer treatment strategies for TNBC with limited treatment options. Both the anti-tumor natural drugs curcumin and ginsenoside Rg3 can exert anti-tumor effects by inducing immunogenic cell death (ICD) of tumor cells, reducing PD-L1 expression, and reducing cancer stem cells. However, they have the disadvantages of poor water solubility, low bioavailability, and weak anti-tumor effect of single agents. We used vinyl ether bonds to link curcumin (Cur) with N-O type zwitterionic polymers and at the same time encapsulated ginsenoside Rg3 to obtain hyperbranched zwitterionic drug-loaded micelles OPDEA-PGED-5HA@Cur@Rg3 (PPH@CR) with pH response. In vitro cell experiments and in vivo animal experiments have proved that PPH@CR could not only promote the maturation of dendritic cells (DCs) and increase the CD4+ T cells and CD8+ T cells by inducing ICD in tumor cells but also reduce the expression of PD-L1 in tumor tissues, and reduce cancer stem cells and showed better anti-tumor effects and good biological safety compared with free double drugs, which is a promising cancer treatment strategy.

Single-cell decoding of drug induced transcriptomic reprogramming in triple negative breast cancers.

BACKGROUND: The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states. RESULTS: We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. CONCLUSIONS: Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration, and re-treatment strategies.

MW-19, a dihydropyrazole derivative, induces human triple-negative breast cancer cell apoptosis by targeting apoptosis-related pathways.

Previous studies have indicated that heterocyclic substituted dihydropyrazole derivatives, particularly MW-19, potentially exert anticancer activity in vitro; however, the underlying mechanism remains unknown. The present study was designed to investigate the mechanisms underlying MW-19 activity in triple-negative breast cancer cells. A sulforhodamine B assay was performed to evaluate cell proliferation inhibition rates, and the antitumor effect of MW-19 was evaluated in mice with HCC-1806 xenografts. Apoptosis was analyzed by Hoechst 33342 and annexin V/propidium iodide staining. Expression of pro- and antiapoptotic proteins and mRNA were analyzed by western blotting and reverse transcription-quantitative (RT-q) PCR, respectively. We found that MW-19 significantly inhibited HCC-1806 cell proliferation in a dose- and time-dependent manner, and significantly inhibited MDA-MB-231 cell migration. Importantly, oral administration of MW-19 significantly inhibited HCC-1806 tumor growth in BALB/c-nu/nu mice. Moreover, MW-19 treatment induced marked apoptosis and G2/M arrest in the sensitive cell line, HCC-1806. RT-qPCR analysis showed that levels of proapoptotic genes (Bax, caspase-3, caspase-7, and Fas) were considerably increased in the MW-19 group relative to the control group, while those of antiapoptotic factors (Bcl-2, C-MYC) were dramatically decreased. Consistently, Bax, caspase-3, and caspase-7 were significantly induced after MW-19 treatment, while levels of phosphorylated (p-)AKT, p-PI3K, p-ERK, and the antiapoptotic protein, Bcl-2, were clearly diminished, and the P38 MAPK signaling pathway was activated. Furthermore, P38 pharmacological inhibitors abrogated MW-19-induced apoptosis. Together, our findings indicate that MW-19 exerts antitumor effects by targeting PI3K/AKT and ERK/P38 signaling pathways.

Fabrication of thiosemicarbazone-based Pd(II) complexes: structural elucidations, catalytic activity towards Suzuki-Miyaura coupling reaction and antitumor activity against TNBC cells.

Currently, there are many uses of metal complexes, especially in the fields of medicinal chemistry and catalysis. Thus, fabrication of new complexes which perform as a catalyst and chemotherapeutic drug is always a beneficial addition to the literature. Herein, we report three heterocyclic thiosemicarbazone-based Pd(II) complexes [Pd(HL1)Cl] (C1), [Pd(L2)(PPh3)] (C2) and [Pd(L3)(PPh3)]Cl (C3) having coligands Cl and PPh3. Thiosemicarbazone ligands (H2L1, H2L2 and HL3) and the complexes (C1-C3) were characterized methodically using several spectroscopic techniques. Single-crystal X-ray diffraction methods reveal that the structural environment around the metal center of C2 is square planar, while for C1 and C3 it is a slighty distorted square plane. The supramolecular network of compounds was built via hydrogen bonds, C-H⋯π and π⋯π interactions. Density functional theory (DFT) study of the structure of the complexes supports experimental findings. The application of these complexes as catalysts toward Suzuki-Miyaura coupling reactions has been examined with various aryl halides and phenyl boronic acid in PEG 400 solvent. The complexes displayed good biomolecular interactions with DNA/protein, with a binding constant value of the order of 105 M-1. C3 showed greater binding efficacy toward these biomolecules than the other complexes, which might be due to the cationic nature of C3. Furthermore, antitumor activity of the complexes was studied against the human triple-negative breast cancer (TNBC) cell line MDA-MB-231. It was found that C3 was more toxic (IC50 = 10 ± 2.90 µM) toward MDA-MB-231 cells than the other complexes. A known chemotherapeutic drug, 5-fluorouracil, was included as positive control. The programmed cell death mechanism of C3 was confirmed. Additionally, complex-induced apoptosis was confirmed and occurred via a mitochondria-dependent (intrinsic) pathway.

Antitumor and antimetastatic effects of dietary sulforaphane in a triple-negative breast cancer models.

Triple-negative breast cancer (TNBC) represents aggressive phenotype with limited treatment options due to the lack of drug targets. Natural compounds are extensively studied regarding their potential to alter the efficacy of cancer treatment Among them sulforaphane - an isothiocyanate of natural origin, was shown to be a hormetic compound, that may exert divergent effects: cytoprotective or cytotoxic depending on its concentrations. Thus, the aim of this study was to determine the effect of its low, dietary concentrations on the proliferation and migration of the TNBC cells in the in vivo and in vitro 2D and 3D model. Results of the in vivo experiment showed up to 31% tumor growth inhibition after sulforaphane treatment associated with lowered proliferating potential of cancer cells, reduced areas of necrosis, and changed immune cell type infiltration, showing less malignant type of tumor in contrast to the non-treated group. Also, the study revealed that sulforaphane decreased the number of lung metastases. The in vitro study confirmed that SFN inhibited cell migration, but only in cells derived from 3D spheroids, not from 2D in vitro cultures. The results show a specific role of sulforaphane in the case of cells released from the TNBC primary tumor and its environment.

Integrated Metabolomics and Transcriptomics Analysis of Anacardic Acid Inhibition of Breast Cancer Cell Viability.

Anacardic acid (AnAc) inhibits the growth of estrogen receptor α (ERα)-positive MCF-7 breast cancer (BC) cells and MDA-MB-231 triple-negative BC (TNBC) cells, without affecting primary breast epithelial cells. RNA sequencing (seq) and network analysis of AnAc-treated MCF-7 and MDA-MB-231 cells suggested that AnAc inhibited lipid biosynthesis and increased endoplasmic reticulum stress. To investigate the impact of AnAc on cellular metabolism, a comprehensive untargeted metabolomics analysis was performed in five independent replicates of control versus AnAc-treated MCF-7 and MDA-MB-231 cells and additional TNBC cell lines: MDA-MB-468, BT-20, and HCC1806. An analysis of the global metabolome identified key metabolic differences between control and AnAc-treated within each BC cell line and between MCF-7 and the TNBC cell lines as well as metabolic diversity among the four TNBC cell lines, reflecting TNBC heterogeneity. AnAc-regulated metabolites were involved in alanine, aspartate, glutamate, and glutathione metabolism; the pentose phosphate pathway; and the citric acid cycle. Integration of the transcriptome and metabolome data for MCF-7 and MDA-MB-231 identified Signal transduction: mTORC1 downstream signaling in both cell lines and additional cell-specific pathways. Together, these data suggest that AnAc treatment differentially alters multiple pools of cellular building blocks, nutrients, and transcripts resulting in reduced BC cell viability.

Cordycepin Enhances the Therapeutic Efficacy of Doxorubicin in Treating Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high mortality and poor prognosis. Meanwhile, doxorubicin, a chemotherapeutic agent for triple-negative breast cancer, has poor sensitivity. The objective of this study was to examine the effect of cordycepin on doxorubicin sensitivity and efficacy in the TNBC xenograft model and explore the relevant molecular pathways. The combination of the drugs in nude mice carrying MDA-MB-231 xenografts significantly reduced the volume, size, and weight of xenografts and improved the tumor inhibition rate. The drug combination was significantly more effective than cordycepin or doxorubicin alone, reflecting the fact that cordycepin enhanced the anti-tumor effects of doxorubicin in MDA-MB-231 xenografts. At the same time, the monitoring of several biological parameters failed to detect any obvious side effects associated with this treatment. After predicting the importance of the TNF pathway in inhibiting tumor growth using network pharmacology methods, we verified the expression of TNF pathway targets via immunohistochemistry and quantitative PCR. Furthermore, a TNF-α inhibitor was able to abrogate the beneficial effects of cordycepin and doxorubicin treatment in MDA-MB-231 cells. This clearly indicates the role of TNF-α, or related molecules, in mediating the therapeutic benefits of the combined treatment in animals carrying TNBC xenografts. The observations reported here may present a new direction for the clinical treatment of TNBC.