Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer.

OBJECTIVE: Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. METHODS: The human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with BNZ-111, and cell proliferation, apoptosis, and cell cycle were assessed. RESULTS: It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating ß-3 tubulin expression to suppress microtubule dynamics. CONCLUSION: BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology.

Long and Short-Term Effect of mTOR Regulation on Cerebral Organoid Growth and Differentiations.

BACKGROUND: The mammalian target of rapamycin (mTOR) signaling is critical for the maintenance and differentiation of neurogenesis, and conceivably for many other brain developmental processes. However, in vivo studies of mTOR functions in the brain are often hampered due to the essential role of the associated signaling in brain development. METHODS: We monitored the long- and short-term effects of mTOR signaling regulation on cerebral organoids growth, differentiation and function using an mTOR inhibitor (everolimus) and an mTOR activator (MHY1485). RESULTS: Short-term treatment with MHY1485 induced faster organoid growth and differentiation, while long-term treatment induced the maturation of cerebral organoids. CONCLUSION: These data suggest that the optimal activity of mTOR is crucial in maintaining normal brain development, and its role is not confined to the early neurogenic phase of brain development.

Synthesis and evaluation of tirbanibulin derivatives: a detailed exploration of the structure-activity relationship for anticancer activity.

Tirbanibulin, an FDA-approved microtubule-targeting agent (MTA) introduced in 2020, represents a pioneering treatment for precancerous actinic keratosis. Despite its failure to gain approval as an anticancer agent due to insufficient efficacy, there remains potential value in extending its application into malignancy treatment through tirbanibulin-based derivatives. Tirbanibulin possesses a distinctive dual mechanism of action involving microtubule and Src inhibition, distinguishing it from other MTAs. In spite of its unique profile, exploration of tirbanibulin's structure-activity relationship (SAR) and the development of its derivatives are significantly limited in the current literature. This study addresses this gap by synthesizing various tirbanibulin derivatives and exploring their SAR through modifications in the core amide motif and the eastern benzylamine part. Our results underscore the critical role of the pyridinyl acetamide core structure for optimal cellular potency, with favorable tolerance observed for modifications at the para position of the benzylamine moiety. Particularly noteworthy is the analogue modified with p-fluorine benzylamine, which exhibited favorable in vivo PK profiles. These findings provide crucial insights into the potential advancement of tirbanibulin-based compounds as promising anticancer agents.

Quantitative and Qualitative Analysis of Neurotransmitter and Neurosteroid Production in Cerebral Organoids during Differentiation.

In the human brain, neurophysiological activity is modulated by the movement of neurotransmitters and neurosteroids. To date, the similarity between cerebral organoids and actual human brains has been evaluated using comprehensive multiomics approaches. However, a systematic analysis of both neurotransmitters and neurosteroids from cerebral organoids has not yet been reported. Here, we performed quantitative and qualitative assessments of neurotransmitters and neurosteroids over the course of cerebral organoid differentiation. Our multiomics approaches revealed that the expression levels of neurotransmitter-related proteins and RNA, including neurosteroids, increase as cerebral organoids mature. We also found that the electrophysiological activity of human cerebral organoids increases in tandem with the expression levels of both neurotransmitters and neurosteroids. Our study demonstrates that the expression levels of neurotransmitters and neurosteroids can serve as key factors in evaluating the maturity and functionality of human cerebral organoids.

Induction of toxicity in human colon cells and organoids by size- and composition-dependent road dust.

Environmental pollution, including the annual resurgence of particulate matter derived from road dust, is a serious issue worldwide. Typically, the size of road dust is less than 10 µm; thus, road dust can penetrate into human organs, including the brain, through inhalation and intake by mouth. Therefore, the toxicity of road dust has been intensively studied in vitro and in vivo. However, in vitro systems, including 2D cell cultures, cannot mimic complex human organs, and there are several discrepancies between in vivo and human systems. Here, we used human colon cells and organoids to evaluate the cytotoxicity of particulate matter derived from road dust. The toxicity of road dust collected in industrialized and high traffic areas and NIST urban particulate matter reference samples were evaluated in 2D and 3D human colon cells as well as colon organoids and their characteristics were carefully examined. Data suggest that the size and elemental compositions of road dust can correlate with colon organoid toxicity, and thus, a more careful assessment of the size and elemental compositions of road dust should be conducted to predict its effect on human health.

A Comparative Systematic Analysis of The Influence of Microplastics on Colon Cells, Mouse and Colon Organoids.

BACKGROUND: Microplastics (MPs) are small fragments from any type of plastic formed from various sources, including plastic waste and microfibers from clothing. MPs degrades slowly, resulting in a high probability of human inhalation, ingestion and accumulation in bodies and tissues. As its impact on humans is a prolonged event, the evaluation of its toxicity and influence on human health are critical. In particular, MPs can enter the human digestive system through food and beverage consumption, and its effect on the human colon needs to be carefully examined. METHODS: We monitored the influence of small MPs (50 and 100 nm) on human colon cells, human colon organoids and also examined their toxicity and changes in gene expression in vivo in a mouse model. RESULTS: The data suggested that 5 mg/mL concentrations of 50 and 100 nm MPs induced a > 20% decrease in colon organoid viability and an increase in the expression of inflammatory-, apoptosis- and immunity-related genes. In addition, in vivo data suggested that 50 nm MPs accumulate in various mouse organs, including the colon, liver, pancreas and testicles after 7 d of exposure. CONCLUSION: Taken together, our data suggest that smaller MPs can induce more toxic effects in the human colon and that human colon organoids have the potential to be used as a predictive tool for colon toxicity.

A screening study of high affinity peptide as molecular binder for AXL, tyrosine kinase receptor involving in Zika virus entry.

The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.

Utilization of road dust chemical profiles for source identification and human health impact assessment.

This study investigated the chemical profiles of fine urban road dust as a set of indicators for major air pollutants at sampling sites or as proxies for potential human health impacts. We examined the chemical compositions of fine particles (< 100 µm) or re-suspended ultrafine particles (< 2.5 µm) in the urban road dust collected from the cities with major emission sources of CO, NH3, NOx, PM2.5, SOx, and volatile organic compounds. The elemental compositions, including metal contents and volatile or semi-volatile organic compound species were determined to constitute comprehensive chemical profiles of the solid road dust samples. The water-extractable organic compounds and fluorescent species of the size-fractionated re-suspended fine particulate matter (RPM) were also incorporated in the chemical profiles. The metal content and aliphatic hydrocarbons could partly distinguish emission sources, and clearer distinctions were achieved with the inclusion of fluorescence excitation-emission matrix (EEM) results. The dose-response test results showed positive correlations between cytotoxicity and relative abundance of hydrocarbons or metal contents of urban road dust. The set of chemical profiles suggested in this study could be further utilized for site identification or human health impact assessment using urban road dust.

Toxicity Assessment of SiO2 and TiO2 in Normal Colon Cells, In Vivo and in Human Colon Organoids.

We conducted systemic assessments on the toxicity of silicon dioxide (SiO2) and titanium dioxide (TiO2) nanoparticles using different forms of normal colon cells (CCD-18Co), in vivo and in human colon organoids. The in vivo acute oral toxicity data showed that the LD50 values are greater than 2000 mg/kg for both the SiO2 and TiO2 nanoparticles; however, the SiO2 and TiO2 nanoparticles induced cytotoxicity in two-dimensional CCD-18Co cells and three-dimensional CCD-18Co spheroids and human colon organoids, with IC50 values of 0.6, 0.8 and 0.3 mM for SiO2 and 2.5, 1.1 and 12.5 mM for TiO2 nanoparticles, respectively. The data suggest that, when SiO2 and TiO2 are in nanoparticle form, cytotoxicity is induced; thus, care should be taken with these materials.

Development of a three-dimensional in vitro co-culture model to increase drug selectivity for humans.

AIM: Insulin resistance is a metabolic state where insulin sensitivity is lower than normal condition and strongly related to type 2 diabetes. However, an in vitro model mimicking insulin resistance is rare and thus screening drugs for insulin resistance severely depends on an in vivo model. Here, to increase anti-diabetic drug selectivity for humans, 3D ADMSCs and macrophages were co-cultured with in-house fabricated co-culture plates. MATERIAL AND METHODS: 3D co-culture plates were designed to load ADMSCs and RAW264.7 cells containing hydrogels in separate wells while allowing cell-cell interaction with co-culturing media. Hydrogels were constructed using a 3D cell-printing system containing 20 mg/ml alginate, 0.5 mg/ml gelatin and 0.5 mg/ml type I collagen. Cells containing hydrogels in 3D co-culture plates were incubated for 10 min to allow stabilization before the experiment. 3D co-culture plates were incubated with the CaCl2 solution for 5 min to complete the cross linking of alginate hydrogel. Cells in 3D co-culture plates were cultured for up to 12 days depending on the experiment and wells containing adipocytes and macrophages were separated and used for assays. RESULTS: KR-1, KR-2 and KR-3 compounds were applied during differentiation (12 days) in 3D co-cultured mouse 3T3-L1 adipocytes and 3D co-cultured human ADMSCs. Glucose uptake assay using 2-DG6P and 2-NBDG and western blot analysis were performed to investigate changes of insulin resistance in the 3D co-cultured model for interspecies selectivity of drug screening. KR-1 (mouse potent enantiomer) and KR-3 (racemic mixture) showed improvement of 2-DG and 2-NBDG uptake compared with KR-2 (human potent enantiomer) in 3D co-cultured 3T3-L1 adipocytes. In connection with insulin resistance in a 3D 3T3-L1 co-cultured model, KR-1 and KR-3 showed improvement of insulin sensitivity compared to KR-2 by markedly increasing GLUT4 expression. In contrast to the result of 3D co-cultured 3T3-L1 adipocytes, KR-1 failed to significantly improve 2-DG and 2-NBDG uptake in 3D co-cultured ADMSC adipocytes. Results of 2-NBDG accumulation and western blot analysis also showed that KR-2 and KR-3 improved insulin sensitivity relatively better than KR-1. CONCLUSIONS: Our 3D co-culture model with/without 3D co-culture plates can successfully mimic insulin resistance while allowing investigation of the effects of anti-obesity or anti-diabetic drugs on human or mouse co-culturing cell type. This 3D co-culture system may accelerate screening of drugs for insulin resistance depending on species.

Purification of Boron Nitride Nanotubes Enhances Biological Application Properties.

Commercially available boron nitride nanotubes (BNNTs) and their purified form (pBNNTs) were dispersed in aqueous solutions with various dispersants, and their cytotoxicity and drug encapsulation capacity were monitored. Our data suggest that pBNNTs showed an average increase in dispersibility of 37.3% in aqueous solution in the presence of 10 different dispersants. In addition, 100 µg of pBNNTs induced an average decrease in cytotoxicity of 27.4% compared to same amount of BNNTs in normal cell lines. The same amount of pBNNTs can encapsulate 10.4-fold more drug (camptothecin) compared to BNNTs. These data suggest that the purification of BNNTs improves several of their properties, which can be applied to biological experiments and are thus essential in the biological application of BNNTs.

Properties of differentiated SH-SY5Y grown on carbon-based materials.

Neural cell differentiation has been extensively studied in two-dimensional (2D) cell culture plates. However, the cellular microenvironment and extracellular matrix (ECM) are much more complex and flat 2D surfaces are hard to mimic in ECM. Carbon nanotubes (CNTs) and graphenes are multidimensional carbon-based nanomaterials and may be able to provide extra dimensions on cell growth and differentiation. To determine the effect of CNTs and graphene surfaces on the growth, gene expression, differentiation and functionality of neuroblastoma to a neural cell, SH-SY5Y cells were grown on a 2D (control) surface, a CNT network and a graphene film. The data suggest that SH-SY5Y cells grown on CNT surfaces show an average 20.2% increase in cell viability; 5.7% decrease in the ratio of cells undergoing apoptosis; 78.3, 43.4 and 38.1% increases in SOX2, GFAP and NeuN expression, respectively; and a 29.7% increase in mean firing rate on a multi-electrode array. SH-SY5Y cells grown on graphene film show little or no changes in cell properties compared to cells grown in 2D. The data indicate that the three-dimensional (3D) surface of CNTs provides a favorable environment for SH-SY5Y cells to proliferate and differentiate to neurons.

Development and structure-activity relationship study of SHP2 inhibitor containing 3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene.

SHP2, a non-receptor protein tyrosine phosphatase encoded by PTPN11 gene, plays an important role in the cell growth and proliferation. Activating mutations of SHP2 have been reported as a cause of various human diseases such as solid tumors, leukemia, and Noonan syndrome. The discovery of SHP2 inhibitor can be a potent candidate for the treatment of cancers and SHP2 related human diseases. Several reports on a small molecule targeting SHP2 have published, however, there are limitations on the discovery of SHP2 phosphatase inhibitors due to the polar catalytic site environment. Allosteric inhibitor can be an alternative to catalytic site inhibitors. 3,4,6-Trihydroxy-5-oxo-5H-benzo[7]annulene 1 was obtained as an initial hit with a 0.097 µM of IC50 from high-throughput screening (HTS) study. After the structure-activity relationship (SAR) study, compound 1 still showed the most potent activity against SHP2. Moreover, compound 1 exerted good potency against SHP2 expressing 2D and 3D MDA-MB-468.

Anticancer effect of XAV939 is observed by inhibiting lactose dehydrogenase A in a 3-dimensional culture of colorectal cancer cells.

XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.

Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using Lactobacillusfermentum for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of Lactobacillus cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.

Two-Photon Probe for TNF-α. Assessment of the Transmembrane TNF-α Level in Human Colon Tissue by Two-Photon Microscopy.

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10-50 cm4s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab-transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.

Long-Term Stability Monitoring of Printed Proteins on Paper-Based Membranes.

Monitoring of long-term stability of proteins on paper-based membranes is important as it is directly related to paper-based sensor fabrication. By using a simple piezo printhead inkjet printer, recombinant proteins and antibodies were printed on paper-based membranes to test their stability and sensitivity under varying lengths of storage and temperature conditions. Our data show that a printed IgG-HRP antibody on simple printing paper maintains >50% functionality up to ∼2 months under 4 and -20 °C storage. Antibodies printed on polyvinylidene difluoride (PVDF) and nitrocellulose showed 5.3 and 9.7% decreases, respectively, in initial signal intensities compared to printing paper. Prostate-specific membrane antigen and tumor necrosis factor alpha recombinant proteins printed on paper-based membranes can be detected by antibodies, and antibody signal intensities can be detected up to 28 days after storage at 4 and -20 °C when printed on PVDF membrane or printing paper. These data suggest that printed proteins on simple printing paper and PVDF membrane can maintain their functionality up to few months when stored at 4 °C or lower and can be potentially applied in paper-based sensor development.

Comparative analysis of urban road dust compositions in relation to their potential human health impacts.

This study investigated the chemical components of fine urban road dust from seven sampling sites, based on which we could predict potential human health effects. The elemental compositions, including the contents of metals and volatile or semivolatile organic compounds, were determined to establish comprehensive chemical profiles of solid road dust. The chemical profiles, consisting of C: H ratio, metal contents, and relative abundances of organic compounds, provided a chemical signature for road dust. To overall cytotoxicity values ranging between 7 and 58%, water extracts contributed less than 15%, and cell death mainly occurred via direct contact with solid-phase components, which possibly indicates that the selected chemical profile of solid-phase road dust components could serve as a strong predictor for BJ and WI-38 cytotoxicity. Pure metal oxides (Cr2O3, CuO, Fe2O3, MnO2, NiO, or ZnO) exhibited a positive dose-response, and the corresponding metal contents in solid road dust were well correlated with cell viability. The principal component analysis (PCA) results suggested that the metal contents were stronger predictors of cytotoxicity than the benzene derivative or hydrocarbon contents. The chemical profiles established in this study could be further utilized to identify candidate health hazard factors in road dust.

αVß3-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture.

Integrin αvß3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvß3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvß3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvß3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.

Effect of fibroblast co-culture on the proliferation, viability and drug response of colon cancer cells.

Interactions between cancer cells and the surrounding fibroblasts serve an important role in cancer proliferation. Colon cancer co-culture model with colon fibroblasts and two metastatic models with lung and skin fibroblasts were established, and the co-culture effects on colon cancer cell proliferation, apoptosis and drug response were evaluated. Co-culture with CCD-18Co and BJ reduces SW480 cell proliferation by 4.2 and 5.3%, respectively, while WI-38 acts as a positive regulator and increases SW480 cell proliferation by 36%. CCD-18Co and BJ co-culture can also enhance XAV939 potency against SW480 cells by 16.8 and 27.3%; however, WI-38 co-culture reduces the effect of XAV939 by 38.2%. The present results suggest that, depending on fibroblast type, co-culture can have a positive/negative influence on colon cancer growth; therefore, care should be taken when considering fibroblasts as a target for future cancer therapies.