Dose-dependent Cell Research Articles

Gabapentin impairs visual development in zebrafish via retinal apoptosis and thyroid disruption.

Gabapentin (GBP), a pharmaceutical widely used for seizures and neuropathic pain, has emerged as a contaminant in global aquatic environments, raising concerns about its ecological impact. This study investigated the effects of environmentally relevant concentrations of GBP (0, 1, 10, 1000 μg/L) on visual development in zebrafish (Danio rerio). Behavioral assays showed that GBP exposure enhanced light sensitivity, as indicated by a significant increase in total travel distance (TTD) in all exposure groups compared to controls. The 1 μg/L and 1000 μg/L exposure groups demonstrated a 41 % and 37 % increase in TTD, respectively (p < 0.05). Apoptosis assays revealed dose-dependent retinal cell death, with fluorescence intensity rising by 15 % at 1000 μg/L (p < 0.05). Visual acuity, measured through optokinetic response (OKR) tests, decreased significantly across all color stimuli. Angular velocity under white light decreased from 4.0 °/s in controls to 1.6 °/s at 1000 μg/L (p < 0.01) in a dose-dependent manner. Retinal histopathology showed a 17 % increase in ganglion cell layer thickness at 1000 μg/L (p < 0.05) in a dose-dependent manner. Thyroid hormone assays indicated significant reductions in T3 and T4 levels (p < 0.001), with a 22 % increase in the T3/T4 ratio at 1000 μg/L. Gene expression analysis revealed dysregulation in apoptosis (casp3a, ifi27), thyroid (tshr, dio1), and retinal development (atoh7, pax6a) pathways. These findings demonstrate that GBP disrupts visual development in zebrafish through retinal apoptosis and thyroid hormone dysregulation, highlighting the ecological risks posed by pharmaceutical pollutants. GBP exposure increased light-driven locomotor activity, indicating heightened light sensitivity due to apoptosis in the retina. Visual acuity was assessed through the optokinetic response (OKR) test, retinal morphology, and thyroid hormone (TH) levels. Even at concentrations as low as 1 µg/L, GBP exposure led to significant reductions in OKR across various colors, likely due to changes in retinal thickness linked to thyroid hormone disruption. These effects were consistent with alterations in gene expression related to apoptosis, the thyroid system, and retinal development. Our findings enhance understanding of how GBP exposure impairs vision in fish and highlight the need to evaluate the ecological risks of pharmaceutical contaminants in aquatic environments.

Effect of surface-engineered AuNPs on gene expression, bacterial interaction, protein denaturation, and toxicology assay: an in vitro and in vivo model.

We investigated the in vitro and in vivo uses of pamoic acid functionalized gold nanoparticles (PA@AuNPs), with a focus on determining their safety and potential toxicity in living beings. To test this theory, the bacterial interaction of PA@AuNPs was studied using Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa cultures, as well as the inhibition of the bovine serum albumin (BSA) protein. The real-time polymerase chain reaction (RT-PCR) is used to measure the expression of target genes. PA@AuNPs caused dose-dependent cell death in MDA-MB-231, a triple-negative breast cancer (BC) cell line, with an LC50 of -42.23 μL mL-1. It also caused apoptosis in BC cells. The results indicated that in the early weeks, inflammatory cells (mostly neutrophils and macrophages) penetrated the connective tissue, but in the latter weeks, a substantial number of fibroblasts and fibrocytes were identified. Changes in vascular channels, extravasated red blood cells (RBCs), and necrosis are all indicators of growing tissue pathology. These data could point to a dynamic process including an anti-inflammatory response followed by tissue remodeling or repair. These findings show that PA@AuNPs were not hazardous to the tested Sprague Dawley rats, are highly biocompatible, and can be used in a variety of biological applications.

Bakuchiol from Cullen corylifolium and its efficacy on apoptosis and autophagy in HepG2 cells

Bakuchiol (4), a component of Cullen corylifolium, has been reported to have estrogenic, antimicrobial, and anti-inflammatory activities. Nonetheless, its anticancer mechanisms and effectiveness against hepatocellular carcinoma remain unexplored. This study sought to elucidate the mechanism of apoptosis, autophagy, and cell cycle arrest caused by bakuchiol (4) and three flavonoids (1-3) with similar structures to compound 4 in hepatocellular carcinoma. Among the evaluated components (1-4), bakuchiol (4) exhibited a significant potential to induce apoptosis in HepG2 cells. This compound facilitates apoptotic processes by engaging both intrinsic and extrinsic signaling cascades, as evidenced by the enhanced ratios of Bax to Bcl-2 and tBid to Bid. In addition, bakuchiol (4) induced a dose-dependent cell cycle arrest, as assessed using a TaliⓇ image-based cytometer. Since bakuchiol decreased CDK2 and CDK4, while increasing p53, p21, and p27, these data suggest that bakuchiol regulated early cell cycle progression. It also promotes the activity of AMPK and the LC3Ⅱ / LC3Ⅰ ratio, while suppressing Akt and mTOR. In conclusion, these results demonstrate that bakuchiol (4), a major component of C. corylifolium, has an anticancer effect in hepatocarcinoma cells by inducing both apoptosis and autophagy. This significant finding enlightens us about the potential of bakuchiol in cancer research, particularly in liver cancer treatment.

Unveiling cadmium toxicity in Etroplus suratensis through endpoint analysis and biomarker profiling: A step towards coastal water quality guideline development

This study contributes to ecotoxicological data essential for establishing water quality standards for cadmium (Cd). The 96-hour median lethal concentration (LC50) of Cd on Etroplus suratensis was 1.56 ± 0.02 mg L−1 with upper and lower confidence intervals of 1.24 to 1.89 mg L−1 respectively. The No Observable Effect Concentration (NOEC), Lowest Observable Effect Concentration (LOEC), and chronic values were 0.11, 0.18, and 0.15 mg L−1, respectively. Antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and total glutathione (TG) increased dose-dependently. Lipid peroxidation (LPO) significantly rose (p < 0.05). Acetylcholine esterase (AChE) activity in the brain and muscle decreased significantly at 0.32 (p < 0.01) and 0.47 (p < 0.001) mg L−1. Energy reserves and electron transport system (ETS) activity dropped in Cd-exposed groups. Cellular energy allocation decreased from controls to the highest exposure group. Behavioral changes such as rubbing, surfacing, jerking, burst swimming, and fin flickering intensified with exposure. Hematological analysis showed a dose-dependent red blood cell (RBC) count decrease and a white blood cell (WBC) count increase. Major gill lesions included lamellar aneurism, lamellar fusion, necrosis, hyperplasia, and hypertrophy. Liver abnormalities featured blood vessel dilation, thrombosis, hemosiderin formation, nuclear pyknosis, and necrosis. Retinular layer damage affected pigment and photoreceptor cells. The integrated biomarker index indicated significant effects on GST, GPx, ETS activity, and LPO content. Also, the high sensitivity of Etroplus suratensis to cadmium confirms its suitability as an indicator species for pollution monitoring.

SURG-58. COLD ATMOSPHERIC PLASMA (CAP) CAUSES SELECTIVE TUMOR CELL DEATH VIA APOPTOSIS AND FERROPTOSIS IN MALIGNANT GLIOMA CELLS

Abstract BACKGROUND High-grade gliomas (HGGs) are primary brain tumors associated with significant morbidity and mortality, and despite advances in neuro-oncology, the prognosis remains poor. Cold atmospheric plasma, a novel experimental anti-cancer therapeutic tool, is a near-room temperature ionized gas generated at atmospheric pressure. It has many promising results in vitro and in vivo with a variety of cancers. CAP cytotoxicity appears to be selective to cancer cells without significant damage to surrounding healthy tissues. We aim to evaluate the efficacy of CAP in generating tumor-selective cytotoxicity against high-grade glioma. METHODS B16, U87, GL261, patient derived high grade glioma, human neural stem cells (LM-NSC008) and astrocytes (NHA hTERT) were treated with CAP. Proliferation and propidium iodide (PI)/annexin V flow cytometry assays were employed to quantify cytotoxicity, cell cycle phases and apoptosis. Cells were pre-treated with ferroptosis inhibitors Ferrostatin-1 and Deferoxamine (DFO) in rescue assays. Cerebral organoids were transplanted with fluorescent tumor cell lines and stained with cleaved caspase-3, ki67 and transferrin receptor. RESULTS CAP induces dose dependent cell death in all glioma cell lines tested. CAP induces an accumulation of intracellular ROS, most prominent in glioma cells. CAP-mediated cell death involves apoptosis and ferroptosis. CAP selectively leads to apoptosis in tumor implanted organoids and leads to decreased proliferation in malignant and non-malignant cells. CAP leads to a shift into G0 phase of all treated cells, malignant and non-malignant. CONCLUSIONS CAP treatment induces dose-dependent increases in ROS and apoptosis in HGG lines tested more significantly than in NHAs and hNSCs. CAP induces decreased proliferation and G0 phase cell cycle arrest in all CAP-treated cells. CAP-mediated cytotoxicity was significantly mitigated with DFO pre-treatment in HGG cells, suggesting that ferroptosis plays a critical role in the mechanism of CAP treatment in HGG. CAP led to selective glioma cell death in tumor implanted cerebral organoids.

CCRG-03. DETECTION OF HETEROGENEOUS CELL CYCLE ALTERATIONS UPON TMZ CHEMOTHERAPY TREATMENT THROUGH ADVANCED MACHINE LEARNING

Abstract Glioblastoma (GB), the most aggressive central nervous system tumor, is hallmarked by its frequent relapse under alkylating chemotherapy. GB cells frequently exhibit changes in cell cycle phases during TMZ treatment, with a reduction in the G0-G1 phase and an increase in the S and G2 phases. This indicate both tumor cell re-entry into the cell cycle and a TMZ-induced arrest, contributing to resistance and progression. In this study, we leverage dynamic protein abundance of Ki-67, a proliferation marker peaking during S, G2, and M phases, to analyse cellular responses to TMZ in various cell lines and tissue samples. We designed an image-based machine learning approach for pattern recognition to reconstruct and analyse the cell cycle at the single-cell level. Six primary cell lines were characterized through whole-genome DNA, methylation and RNA sequencing. Functional read-outs were performed with large-scale, time- and concentration-dependent TMZ treatments in cell culture and GB-tissue slice models. Our model successfully reconstructed detailed cell cycle stages, showing that untreated cells were predominantly in the G0-G1 phase. TMZ treatment significantly shifted cells toward the S/G2 phase (p &amp;lt; 0.02) in 3 of 6 cell lines and the pseudo-time reconstruction highlighted dose-dependent cell cycle changes. Cell lines with enhanced stem-like features, defined based on epigenetic and transcriptional data signature, showed no cell cycle changes at high TMZ doses (&amp;gt;150µM), regardless the MGMT promoter methylation status. This heterogeneity in cell cycle response emphasizes the complexity of GB and the need for personalized treatments. Our study provides insights into GB MGMT cell cycle alterations and resistance mechanisms that are independent of MGMT methylation status, a known TMZ response biomarker. Therefore, the observed TMZ-induced cell cycle changes may serve as novel biomarkers for assessing treatment response, paving the way for personalized therapeutic approaches.

Pyroptosis is not likely to play a major role in anthracycline-induced cytotoxicity in H9c2 cardiomyocytes and human cardiac endothelial cells

Abstract Background Although anthracyclines are widely used as effective chemotherapeutic agents, anthracycline-induced cardiotoxicity (AIC) causes significant morbidity and mortality. The precise mechanism of AIC remains elusive, although apoptosis may be involved. Recent research suggests pyroptosis, a programmed lytic cell death pathway, might also contribute to AIC. Although studies have focused on AIC in the myocardium, emerging evidence suggests that AIC may also affect vascular cells. Furthermore, the cytotoxicity of doxorubicin (DOX) or its supposed active metabolite, doxorubicinol (DOXOL), has not been widely investigated in cardiac vascular cells. Purpose This study examines the hypothesis that pyroptosis plays a role in DOX and DOXOL cytotoxicity in H9c2 immortalised rat cardiac myoblasts and two types of human cardiac endothelial cells. Methods In vitro experiments were conducted to ascertain dose-dependent toxicity in H9c2, and cardiac endothelial cells following treatment with DOX or DOXOL. The type of cell death was determined by assessing cell morphology and fluorescent staining with markers of apoptosis (Annexin V) and lytic cell death (Propidium Iodide PI) following DOX or DOXOL treatment in comparison to known inducers of pyroptosis (LPS &amp; Nigericin). Western blot experiments were conducted to study the expression of GSDMD (involved in pyroptosis), and caspase 3 (involved in apoptosis), following DOX or DOXOL. A human monocytic acute myeloid leukaemia cell line (THP-1 cells) was used as a positive control for pyroptosis. Results Dose-dependent cell death following DOX treatment was higher than with DOXOL in both H9c2 and cardiac endothelial cells. DOX caused apoptotic changes in all cell types. In contrast, DOXOL had weaker effects, inducing early apoptosis. No cleavage (activation) of GSDMD or caspase 3 was detected after DOX or DOXOL. Interestingly, in THP-1 cells, DOX induced predominantly apoptotic cell death, whereas DOXOL induced predominantly pyroptotic cell death, assessed both morphologically and by caspase 3 and GSDMD cleavage. Conclusion DOX and DOXOL primarily induce apoptotic cell death in H9c2 cardiomyocytes and cardiac endothelial cells. Interestingly, DOXOL, supposedly the active metabolite, was less cytotoxic than DOX. THP-1 cells undergo apoptotic or pyroptotic cell death with DOX or DOXOL, respectively. In conclusion, anthracycline-induced cytotoxicity is likely to be due to apoptotic injury rather than pyroptosis.

Methyl Gallate Suppresses Canine Mammary Gland Tumors by Inducing Apoptosis and Anti-angiogenesis.

Methyl gallate (MG), a plant phenolic compound, has known anticancer properties. However, its effects on canine mammary gland tumors (CMTs) are unclear. This study evaluated the impact of MG on cell viability, migration, and apoptosis in two CMT cell lines. CMT-U27 and CF41.mg cells were used. In vitro experiments included MTT and scratch assays, Annexin-V/propidium iodide double staining, immunocytochemistry, and western blot analyses. An in vivo CMT xenograft mouse model was also used to observe the effects of MG on tumor growth and vasculature. Immunohistochemistry was performed to analyze vessel density and apoptosis in tumor tissues. Cell migration and tube formation assays with canine aortic endothelial cells assessed the anti-angiogenic effects of MG. Data showed a significant decrease in cell viability and migration in both CMT cell lines after 24 h exposure to various MG concentrations. MG treatment induced dose-dependent apoptotic cell death and elevated cleaved caspase-3 expression. In vivo experiments confirmed tumor growth suppression 21 days post-treatment with 40 mg/kg MG. Tumor tissues displayed increased cleaved caspase-3 and reduced vessel density. MG also inhibited cell migration and disrupted tube formation in canine endothelial cells. MG has potential as an anticancer drug for CMTs by promoting apoptotic cell death and reducing angiogenesis, highlighting its therapeutic promise.

Design and development of Fujicalin-based axitinib liquisolid compacts for improved dissolution and bioavailability to treat renal cell carcinoma

Poor dissolution of axitinib (AXT) limits its effectiveness through the oral route. The present study investigated, prospective of liquisolid (LS) technology to improve dissolution rate and oral bioavailability of AXT to treat renal cell carcinoma. LS compacts were fabricated with PEG 200, Fujicalin SG, and Aerosil 200 as solvent, carrier, and coat material, respectively. The behavior of LS-systems during tabletting was investigated using Kawakita, Heckel, and Leuenberger analysis. LS compacts were examined for P-XRD, DSC, SEM, and in vitro drug dissolution. For optimization, a 32 full factorial design was utilized. Cell line A498 was utilized for in vitro cytotoxicity study. A bioavailability study was performed using rabbits. DSC and P-XRD analysis confirmed the transition of crystalline AXT to its partial amorphization and molecular dispersion. Consequently, LS6 demonstrated a significantly rapid drug dissolution (Q20; >99 %) than the directly compressed tablets (18.05 %). Additionally, 2.03-fold increase in oral bioavailability, and inhibited dose-dependent cell growth with 1.75-fold increased apoptosis rate. Overall, an LS6 compact consisting of 15 % AXT concentration in PEG 200 and a 20 w/w ratio of Fujicalin SG: Aerosil 200 exhibited improved formulation properties, enhanced dissolution rate, and bioavailability. Thus developed potential product may contribute low-cost production with patient-improved survival expectations.

Ameliorative effects of silymarin on aflatoxin B1 toxicity in weaned rabbits: impact on growth, blood profile, and oxidative stress

Natural plant extracts offer numerous health benefits for rabbits, including improved feed utilization, antimycotic and antiaflatoxigenic effect, antioxidants, immunological modulation, and growth performance. The aim of the current study was to investigate the effects of silymarin on the performance, hemato-biochemical indices, antioxidants, and villus morphology. A total of 45 Moshtohor 4 weeks old weaned male rabbits were randomly allocated into three groups (15 rabbit/each) each group with 5 replicates. The first group served as the control group feed on an infected diet by aflatoxin B1 (AFB1) 0.02 mg/kg BW, while the second and third groups received an infected diet by AFB1 (0.02 mg/kg BW) and was treated with Silymarin 20 mg/kg BW/day or 30 mg/kg BW/day, respectively. Regarding the growth performance, silymarin supplementation significantly improved the final body weight compared with the control group. Physiologically, silymarin induced high level of dose-dependent total red blood cell count, hematocrit, eosinophils, high-density lipoprotein cholesterol, superoxid dismutase, catalase activity, total antioxidant capacityand intestinal villi width and length. Moreover, silymarin significantly restricted oxidative stress indicators, malondialdehyde, Alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceridein rabbits treated with (AFB1). In conclusion, silymarin supplementation to AFB1 contaminated rabbit diet may mitigate the negative effect of AFB1 on the rabbit performance and health status and increase growth performance, average daily gain, immunological modulation and antioxidants and provide a theoretical basis for the application of silymarin in livestock production.

Enhanced Antibacterial Activity of Carbon Dots: A Hybrid Approach with Levofloxacin, Curcumin, and Tea Polyphenols

Bacterial infections and their increasing resistance to antibiotics pose a significant challenge in medical treatment. This study presents the synthesis and characterization of novel carbon dots (CDs) using levofloxacin (Lf), curcumin (Cur), and tea polyphenols (TP) through a facile hydrothermal method. The synthesized curcumin-tea polyphenol@carbon dots (Cur-TP@CDs) and levofloxacin-tea polyphenol@carbon dots (Lf-TP@CDs) were characterized using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy, confirming their unique structural and chemical properties. Cur-TP@CDs exhibited an average particle size of 1.32 nanometers (nm), while Lf-TP@CDs averaged 1.58 nm. Both types demonstrated significant antibacterial activity, with Lf-TP@CDs showing superior effectiveness against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in broth dilution and disc diffusion assays. Biofilm inhibition assays revealed a significant reduction in biofilm formation at higher concentrations. The ultraviolet-visible (UV-vis) and photoluminescence (PL) spectral analyses indicated efficient photon emission, and electron paramagnetic resonance (EPR) analysis showed increased singlet oxygen generation, enhancing bactericidal effects. Live and dead bacterial staining followed by scanning electron microscopy (SEM) analysis confirmed dose-dependent bacterial cell damage and morphological deformities. These findings suggest that Cur-TP@CDs and Lf-TP@CDs are promising antibacterial agents, potentially offering a novel approach to combat antibiotic-resistant bacterial infections.

Copper ions-photo dual-crosslinked alginate hydrogel for angiogenesis and osteogenesis.

Early healing of bone defects is still a clinical challenge. Many bone-filling materials have been studied, among which photocrosslinked alginate has received significant attention due to its good biocompatibility and morphological plasticity. Although it has been confirmed that photocrosslinked alginate can be used as an extracellular matrix for 3D cell culture, it lacks osteogenesis-related biological functions. This study constructed a copper ions-photo dual-crosslinked alginate hydrogel scaffold by controlling the copper ion concentration. The scaffolds were shaped by photocrosslinking and then endowed with biological functions by copper ions crosslinking. According to in vitro research, the dual-crosslinked hydrogel increased the compressive strength and favored copper dose-dependent osteoblast differentiation and cell surface adherence of rat bone marrow mesenchymal stem cells and the expression of type I collagen (Col1), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), vascular endothelial growth factor (VEGF). In addition, hydrogel scaffolds were implanted into rat skull defects, and more angiogenesis and osteogenesis could be observed in in vivo studies. The above results show that the copper-photo-crosslinked hydrogel scaffold has excellent osseointegration properties and can potentially promote angiogenesis and early healing of bone defects, providing a reference solution for bone tissue engineering materials.

Thymoquinone reversed doxorubicin resistance in U87 glioblastoma cells via targeting PI3K/Akt/mTOR signaling.

Natural compounds such as thymoquinone (TQ) have recently gained increasing attention in treating glioblastoma (GBM). However, the effects of TQ in reversing drug resistance are not completely understood. Therefore, we aimed to examine TQ impacts on GBM cells with doxorubicin (DOX) resistance and the involvement of the PI3K/Akt/mTOR pathway. GBM cancer U87 and U87/DOX (resistant cells) cells were exposed to DOX and TQ, and cell proliferation was assessed by the MTT assay. ELISA was applied to evaluate cell apoptosis. The expression of apoptotic mediators such as Caspase-3, Bax, Bcl-2 and PI3K, Akt, mTOR, P-gp, and PTEN was assessed via qRT-PCR and western blot. We found that a combination of TQ and DOX suppressed dose-dependent cell growth capacity in cells and increased the cytotoxic effects of DOX in resistant cells. In addition, TQ treatment increased DOX-mediated apoptosis in U87/DOX cell lines via modulating the pro- and anti-apoptotic markers. A combination of TQ and DOX upregulated PTEN and downregulated PI3K, Akt, and mTOR, suppressing this signal transduction in resistant cells. In conclusion, we showed TQ potentiated doxorubicin-mediated antiproliferative and pro apoptotic function DOX-resistant glioblastoma cells, which is mediated by targeting and suppressing PI3K/Akt/mTOR signal transduction.

Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

Xc- System as a Possible Target for ConBr Lectin Interaction in Glioma Cells

Studies have revealed the dependence of glioma cells on iron, making them sensitive to ferroptosis. Ferroptosis can be triggered by inhibition of the xc- system, resulting in redox imbalance and membrane lipid peroxidation. The xc- system is composed of two coupled proteins, xCT and CD98hc. The control of transporters, such as xCT, by the CD98hc glycoprotein suggests that molecules targeting glycans may have an impact on the treatment of glioma. This study evaluated the effect of the Canavalia brasiliensis (ConBr) lectin on C6 glioma cells and compared it with erastin, an xc- system inhibitor. Both induced dose-dependent cell death, accompanied by an increase in the production of reactive oxygen species and a decrease in reduced glutathione. However, co-treatment did not show an additive effect. The analysis was updated by molecular dynamics assessments of the xc- system interacting with ConBr or erastin. The interaction of erastin with the xc- system affects its interaction with ConBr, reducing the antagonistic effect when both are in the protein complex. The data show that ConBr is effective in inducing cell death in glioma cells and regulates the xc system through interaction with CD98hc glycans, showing that lectins have the potential to promote ferroptosis in glioma cells.

Sulfated Polyhydroxysteroid Glycosides from the Sea of Okhotsk Starfish Henricia leviuscula spiculifera and Potential Mechanisms for Their Observed Anti-Cancer Activity against Several Types of Human Cancer Cells.

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A (1), B (2), and C (3), along with new related unsulfated monoglycoside, spiculiferoside D (4), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1-3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides (2, 3) are biosides, and one glycoside (1), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO3-α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1-3 (at concentrations up to 100 μM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

Abstract PR001: KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling synthetic lethality target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX020, inhibits KIF18A ATPase activity with an IC50 of 0.014 µM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 &amp;gt; 10 µM) and EG5 (IC50 5.87 µM). ATX020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 0.053 µM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In these sensitive cell lines, ATX020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX020 exhibit fragmented nuclei and malformed mitotic spindles. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX020 treatment, cell division is unaffected, and no induction of Annexin V, phospho-Histone H3, and gamma-H2AX is observed. This selective dependency is also observed in vivo, where administration of ATX020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Laura Ghisolfi, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR001.

Transdermal peptide delivery using solvent-free thermosponge nanoparticles to improve the anti-aging efficacy of peptides in clinical trials

With the worldwide increase in life expectancy, the human desire to maintain youth and beauty has intensified, leading to rapid advances in the cosmetic industry. However, recently proposed peptide-based formulations with anti-aging properties exhibit low skin-penetration ability. To overcome this limitation, an anti-wrinkle peptide (Regentide®-AWP013; RG) with time- and dose-dependent cell proliferation, wound-healing activity, and no cytotoxicity was developed in this study. The stability and efficacy of RG were improved using solvent-free thermosponge nanoparticles (TNPs) comprising FDA-approved biocompatible materials, which were developed using a simple nanoprecipitation method. The temperature-responsive TNPs efficiently encapsulated RG in aqueous solutions (without any organic solvents), thus improving the cellular uptake and skin-penetration ability of RG. The loading content of RG in TNPs was optimized by monitoring the changes in the physicochemical properties of the RG-loaded TNPs (RG@TNP). The optimally loaded RG@TNP showed long-term stability under physiological conditions, with better skin-penetration ability than free RG. In clinical trials, the RG@TNP showed excellent anti-aging effects on eye wrinkles; it improved skin elasticity and reduced skin pigmentation and sagging, indicating a massive potential in the cosmetic industry. The results obtained in this study confirm the ability of TNPs to efficiently encapsulate peptides with anti-wrinkle properties that improve their stability and skin-penetration ability.

Natural products to enhance anti-tumor effects of trastuzumab and paclitaxel on the SKBR3, which is human breast cancer cell line.

e13051 Background: Neoadjuvant chemotherapy (ChT) with targeted therapy is the standard treatment for human epidermal growth factor 2 (HER2)- positive breast cancer (BC). In this context, Trastuzumab (Tz) and ChT significantly improved the pathological complete response (pCR) rate. Tz and Paclitaxel (PTX) combination therapy is also one of the neoadjuvant treatments used in HER2+BC, and despite the effectiveness of this treatment, there is a critical need for innovative approaches and further research that will improve the neoadjuvant response rate. Addressing this, the NaturaProDB tool presents a novel avenue for predicting in silico natural non-toxic compounds with potential anti-cancer properties. Our study aims to investigate synergistic effect of natural products proposed by this database on theurapeutic action of Tz and PTX on BC. Methods: The NaturaProDB database was searched using the ‘Target Cancer’ option in order to identify those natural products with anti-BC potential and the corresponding experimentally verified target genes. Using this database, Biochanin A (BCA) and Delphinidin (Dp) were selected as natural compounds because they both commonly inhibit the MAPK signaling pathway. We used the HER2+ SKBR3 cell line to determine IC20, IC30 and IC50 doses for BCA, Dp, Tz, and PTX through MTT assay. Notably, all experiments were done after 72 hrs treatment of all compounds. Further, various combinations within different concentrations of natural products and Tz and PTX were tested to define additive anti-proliferative effects on SKBR3 cell line. Results: We first defined two natural products with drug-like properties, BCA and Dp, which have anti-proliferative effect on BC. Treatment of SKBR3 with increasing concentrations of BCA and Dp caused dose-dependent proliferation inhibition and IC50 dose of compounds were defined as 42,70uM and 40,70uM, respectively. In addition, IC20 dose of Tz was 2,8µM, while IC30 dose of PTX was 2,3nM.Then, we showed that combinations of increasing doses of Dp (from 30µM to 90 µM) with IC20 Tz caused significant dose-dependent cell death compared to the control group (p&lt;0.0001). And the combinations of increasing doses of BCA (from 45µM to 360 µM) with IC20 Tz led similar significant results (p&lt;0.0001). Conclusions: Herein, it is shown that combining natural compounds (Dp, BCA) with conventional treatments (PTX,Tz) predicted for the SKBR3 cell line can enhance the inhibitory effect on cell proliferation. This study is one of the rare first cell line studies in which the synergistic effect of BCA and Dp, which are natural compounds selected using the NaturaProDB database, were combined with ChT and Tz to show their synergistic effect, and these findings indicate that synergistic combination treatments acting through different pathways (MAPK signaling pathway) can be used in the treatment of HER2+ and TN BC. It opens avenues for further research.

Novel fabrication of anti-VEGF drug ranibizumab loaded PLGA/PLA co-polymeric nanomicelles for long-acting intraocular delivery in the treatment of age-related macular degeneration therapy

Age associated macular degeneration is the 3rd primary cause of blind fundus diseases globally. A reliable and long-lasting method of intraocular drug delivery is still needed. Herein, this study was aim to develop the novel fabrication of ranibizumab loaded co-polymeric nanomicelles (Rabz-CP-NMs) for AMD. The CMC of co-polymeric nanomicelles was determined to be low, at 6.2μg/ml. The ring copolymerization method was employed to fabricate the NMs and characterize via FTIR, XRD, TEM, DLS and Zeta potential. Rabz-CP-NMs was spherical shape with 10-50nm in size. Stable and prolonged drug release was achieved with the Rabz from CP-NMs at 48h. D407 and ARPE19 ocular cell lines showed dose-dependent cell viability with Rabz-CP-NMs. The Rabz-CP-NMs also had less toxicity, higher uptake, lower cell death and prolonged VEGF-A inhibition, as shown by cytoviability assay. Thus, Rabz-CP-NMs were safe for ocular use, suggesting that could be used to improve intraocular AMD treatment.