Review: Human stem cell-based 3D in vitro angiogenesis models for preclinical drug screening applications.

Vascular diseases are the underlying pathology in many life-threatening illnesses. Human cellular and molecular mechanisms involved in angiogenesis are complex and difficult to study in current 2D in vitro and in vivo animal models. Engineered 3D in vitro models that incorporate human pluripotent stem cell (hPSC) derived endothelial cells (ECs) and supportive biomaterials within a dynamic microfluidic platform provide a less expensive, more controlled, and reproducible platform to better study angiogenic processes in response to external chemical or physical stimulus. Current studies to develop 3D in vitro angiogenesis models aim to establish single-source systems by incorporating hPSC-ECs into biomimetic extracellular matrices (ECM) and microfluidic devices to create a patient-specific, physiologically relevant platform that facilitates preclinical study of endothelial cell-ECM interactions, vascular disease pathology, and drug treatment pharmacokinetics. This review provides a detailed description of the current methods used for the directed differentiation of human stem cells to endothelial cells and their use in engineered 3D in vitro angiogenesis models that have been developed within the last 10 years.

Novel Unsymmetric 3,5-Bis(benzylidene)-4-piperidones That Display Tumor-Selective Toxicity.

Two series of novel unsymmetrical 3,5-bis(benzylidene)-4 piperidones 2a-f and 3a-e were designed as candidate antineoplastic agents. These compounds display potent cytotoxicity towards two colon cancers, as well as several oral squamous cell carcinomas. These compounds are less toxic to various non-malignant cells giving rise to large selectivity index (SI) figures. Many of the compounds are also cytotoxic towards CEM lymphoma and HL-60 leukemia cells. Representative compounds induced apoptotic cell death characterized by caspase-3 activation and subG1 accumulation in some OSCC cells, as well as the depolarization of the mitochondrial membrane potential in CEM cells. A further line of inquiry was directed to finding if the SI values are correlated with the atomic charges on the olefinic carbon atoms. The potential of these compounds as antineoplastic agents was enhanced by an ADME (absorption, distribution, metabolism, and excretion) evaluation of five lead molecules, which revealed no violations.

An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.

Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial ß-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.

A Novel Pyrazole Exhibits Potent Anticancer Cytotoxicity via Apoptosis, Cell Cycle Arrest, and the Inhibition of Tubulin Polymerization in Triple-Negative Breast Cancer Cells.

In this study, we screened a chemical library to find potent anticancer compounds that are less cytotoxic to non-cancerous cells. This study revealed that pyrazole PTA-1 is a potent anticancer compound. Additionally, we sought to elucidate its mechanism of action (MOA) in triple-negative breast cancer cells. Cytotoxicity was analyzed with the differential nuclear staining assay (DNS). Additional secondary assays were performed to determine the MOA of the compound. The potential MOA of PTA-1 was assessed using whole RNA sequencing, Connectivity Map (CMap) analysis, in silico docking, confocal microscopy, and biochemical assays. PTA-1 is cytotoxic at a low micromolar range in 17 human cancer cell lines, demonstrating less cytotoxicity to non-cancerous human cells, indicating a favorable selective cytotoxicity index (SCI) for the killing of cancer cells. PTA-1 induced phosphatidylserine externalization, caspase-3/7 activation, and DNA fragmentation in triple-negative breast MDA-MB-231 cells, indicating that it induces apoptosis. Additionally, PTA-1 arrests cells in the S and G2/M phases. Furthermore, gene expression analysis revealed that PTA-1 altered the expression of 730 genes at 24 h (198 upregulated and 532 downregulated). A comparison of these gene signatures with those within CMap indicated a profile similar to that of tubulin inhibitors. Subsequent studies revealed that PTA-1 disrupts microtubule organization and inhibits tubulin polymerization. Our results suggest that PTA-1 is a potent drug with cytotoxicity to various cancer cells, induces apoptosis and cell cycle arrest, and inhibits tubulin polymerization, indicating that PTA-1 is an attractive drug for future clinical cancer treatment.

A photothermal driven chemotherapy for the treatment of metastatic melanoma.

Solid tumors are abnormal mass of tissue, which affects the organs based on its malignancy and leads to the dysfunction of the affected organs. The major problem associated with treatment of solid tumors is delivering anticancer therapeutics to the deepest layers/core of the solid tumor. Deposition of excessive extracellular matrix (ECM) hinders the therapeutics to travel towards the core of the tumor. Therefore, conventional anticancer therapeutics can only reduce the tumor size and that also for a limited duration, and tumor recurrence occurs once the therapy is discontinued. Additionally, by the time the cancer is diagnosed, the cancer cells already started affecting the major organs of the body such as lung, liver, spleen, kidney, and brain, due to their ability to metastasize and lung is the primary site for them to be infiltrated. To facilitate the anticancer therapeutics to penetrate the deeper layers of tumor, and to provide concurrent treatment of both the solid tumor and metastasis, we have designed and developed a Bimodal Light Assisted Skin Tumor and Metastasis Treatment (BLAST), which is a combination of photothermal and chemotherapeutic moieties. The BLAST is composed of 2D boron nitride (BN) nanosheet with adsorbed molecules of BCL-2 inhibitor, Navitoclax (NAVI) on its surface, that can breakdown excessive ECM network and thereby facilitate dissociation of the solid tumor. The developed BLAST was evaluated for its ability to penetrate solid tumors using 3D spheroids for the uptake, cytotoxicity, growth inhibition, reactive oxygen species (ROS) detection, penetration, and downregulation of proteins upon laser irradiation. The in vivo therapeutic studies on a skin cancer mice model revealed that the BLAST with and without laser were able to penetrate the solid tumor, reduce tumor volume in mice, dissociate the protein network, and prevent lung metastasis as confirmed by immunohistochemistry and western blot analysis. Post analysis of serum and blood components revealed the safety and efficacy of BLAST in mice. Hence, the developed BLAST holds strong promise in solid tumor treatment and metastasis prevention simultaneously.

Topical Administration of an Apoptosis Inducer Mitigates Bleomycin-Induced Skin Fibrosis.

Pathological fibrosis is distinguished from physiological wound healing by persistent myofibroblast activation, suggesting that therapies that induce myofibroblast apoptosis selectively could prevent progression and potentially reverse the established fibrosis, such as for scleroderma (a heterogeneous autoimmune disease characterized by multiorgan fibrosis). Navitoclax (NAVI) is a BCL-2/BCL-xL inhibitor with antifibrotic properties and has been investigated as a potential therapeutic for fibrosis. NAVI makes myofibroblasts particularly vulnerable to apoptosis. However, despite NAVI's significant potency, clinical translation of BCL-2 inhibitors, NAVI in this case, is hindered due to the risk of thrombocytopenia. Therefore, in this work, we utilized a newly developed ionic liquid formulation of NAVI for direct topical application to the skin, thereby avoiding systemic circulation and off-target-mediated side effects. The ionic liquid composed of choline and octanoic acid (COA) at a 1:2 molar ionic ratio increases skin diffusion and transportation of NAVI and maintains their retention within the dermis for a prolonged duration. Topical administration of NAVI-mediated BCL-xL and BCL-2 inhibition results in the transition of myofibroblast to fibroblast and ameliorates pre-existing fibrosis, as demonstrated in a scleroderma mouse model. We have observed a significant reduction of α-SMA and collagen, which are known as fibrosis marker proteins, as a result of the inhibition of anti-apoptotic proteins BCL-2/BCL-xL. Overall, our findings show that COA-assisted topical delivery of NAVI upregulates apoptosis specific to myofibroblasts, with minimal presence of the drug in the systemic circulation, resulting in an accelerated therapeutic effect with no discernible drug-associated toxicity.

Three novel piperidones exhibit tumor-selective cytotoxicity on leukemia cells via protein degradation and stress-mediated mechanisms.

BACKGROUND: Cancer is an ongoing worldwide health problem. Although chemotherapy remains the mainstay therapy for cancer, it is not always effective and has detrimental side effects. Here, we present piperidone compounds P3, P4, and P5 that selectively target cancer cells via protein- and stress-mediated mechanisms. METHODS: We assessed typical apoptotic markers including phosphatidylserine externalization, caspase-3 activation, and DNA fragmentation through flow cytometry. Then, specific markers of the intrinsic pathway of apoptosis including the depolarization of the mitochondria and the generation of reactive oxygen species (ROS) were investigated. Finally, we utilized western blot techniques, RT-qPCR, and observed the cell cycle profile after compound treatment to evaluate the possible behavior of these compounds as proteasome inhibitors. For statistical analyses, we employed the one-way ANOVA followed by Bonferroni post hoc test. RESULTS: P3, P4, and P5 induce cytotoxic effects towards tumorigenic cells, as opposed to non-cancerous cells, at the low micromolar range. Compound treatment leads to the activation of the intrinsic pathway of apoptosis. The accumulation of poly-ubiquitinated proteins and the pro-apoptotic protein Noxa, both typically observed after proteasome inhibition, occurs after P3, P4, and P5 treatment. The stress-related genes PMAIP1, ATF3, CHAC1, MYC, and HMOX-1 were differentially regulated to contribute to the cytotoxic activity of P3-P5. Finally, compound P5 causes cell cycle arrest at the G2/M phase. CONCLUSION: Taken together, compounds P3, P4, and P5 exhibit strong potential as anticancer drug candidates as shown by strong cytotoxic potential, activation of the intrinsic pathway of apoptosis, and show typical proteasome inhibitor characteristics.

Ionic liquid-mediated delivery of a BCL-2 inhibitor for topical treatment of skin melanoma.

Skin melanoma is one of the most common cancer types in the United States and worldwide, and its incidence continues to grow. Primary skin melanoma can be removed surgically when feasible and if detected at an early stage. Anti-cancer drugs can be applied topically to treat skin cancer lesions and used as an adjunct to surgery to prevent the recurrence of tumor growth. We developed a topical formulation composed of Navitoclax (NAVI), a BCL-2 inhibitor that results in apoptosis, and an ionic liquid of choline octanoate (COA) to treat early-stage melanoma. NAVI is a small hydrophobic molecule that solubilizes at 20% (w/v) when dissolved in 50% COA. Although NAVI is a highly effective chemotherapeutic, it is equally thrombocytopenic. We found that COA-mediated topical delivery of NAVI enhanced its penetration into the skin and held the drug in the deeper skin layers for an extended period. Topical delivery of NAVI produced a higher cancer-cell killing efficacy than orally administrated NAVI. In vivo experiments in a mouse model of human melanoma-induced skin cancer confirmed the formulation's effectiveness via an apoptotic mechanism without any significant skin irritation or systemic absorption of NAVI. Overall, this topical approach may provide a safe and effective option for better managing skin cancer in the clinic.

Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Design, Syntheses, and Bioevaluations of Some Novel N2-Acryloylbenzohydrazides as Chemostimulants and Cytotoxic Agents.

A series of novel N2-acryloylhydrazides 1a-m and a related series of compounds 6a-c were prepared as potential chemostimulants. In general, these compounds are cytotoxic to human HCT 116 colon cancer cells, as well as human MCF-7 and MDA-MB-231 breast cancer cell lines. A representative compound N1-(3,4-dimethoxyphenylcarbonyl)-N2-acryloylhydrazine 1m sensitized HCT 116 cells to the potent antineoplastic agent 3,5-bis(benzylidene)-4-piperidone 2a, and also to 5-fluorouracil. A series of compounds was prepared that incorporated some of the molecular features of 2a and related compounds with various N2-acryloylhydrazides in series 1. These compounds are potent cytotoxins. Two modes of action of representative compounds are the lowering of mitochondrial membrane potential and increasing the concentration of reactive oxygen species.

Ni-Cu Nanoparticles and Their Feasibility for Magnetic Hyperthermia.

Ni-Cu nanoparticles have been synthesized by reducing Ni and Cu from metal precursors using a sol-gel route followed by annealing at 300 °C for 1, 2, 3, 6, 8, and 10 h for controlled self-regulating magnetic hyperthermia applications. Particle morphology and crystal structure revealed spherical nanoparticles with a cubic structure and an average size of 50, 60, 53, 87, and 87 nm for as-made and annealed samples at 300 °C for 1, 3, 6, and 10 h, respectively. Moreover, hysteresis loops indicated ferromagnetic behavior with saturation magnetization (Ms) ranging from 13-20 emu/g at 300 K. Additionally, Zero-filed cooled and field cooled (ZFC-FC) curves revealed that each sample contains superparamagnetic nanoparticles with a blocking temperature (TB) of 196-260 K. Their potential use for magnetic hyperthermia was tested under the therapeutic limits of an alternating magnetic field. The samples exhibited a heating rate ranging from 0.1 to 1.7 °C/min and a significant dissipated heating power measured as a specific absorption rate (SAR) of 6-80 W/g. The heating curves saturated after reaching the Curie temperature (Tc), ranging from 30-61 °C within the therapeutic temperature limit. An in vitro cytotoxicity test of these Ni-Cu samples in biological tissues was performed via exposing human breast cancer MDA-MB231 cells to a gradient of concentrations of the sample with 53 nm particles (annealed at 300 °C for 3 h) and reviewing their cytotoxic effects. For low concentrations, this sample showed no toxic effects to the cells, revealing its biocompatibility to be used in the future for in vitro/in vivo magnetic hyperthermia treatment of cancer.