Non-tumorigenic Epithelial Cells Research Articles

Cancer cell-selective induction of mitochondrial stress and immunogenic cell death by PT-112 in human prostate cell lines

PT-112 is a novel immunogenic cell death (ICD)-inducing small molecule currently under Phase 2 clinical development, including in metastatic castration-resistant prostate cancer (mCRPC), an immunologically cold and heterogeneous disease state in need of novel therapeutic approaches. PT-112 has been shown to cause ribosome biogenesis inhibition and organelle stress followed by ICD in cancer cells, culminating in anticancer immunity. In addition, clinical evidence of PT-112-driven immune effects has been observed in patient immunoprofiling. Given the unmet need for immune-based therapies in prostate cancer, along with a Phase I study (NCT#02266745) showing PT-112 activity in mCRPC patients, we investigated PT-112 effects in a panel of human prostate cancer cell lines. PT-112 demonstrated cancer cell selectivity, inhibiting cell growth and leading to cell death in prostate cancer cells without affecting the non-tumorigenic epithelial prostate cell line RWPE-1 at the concentrations tested. PT-112 also caused caspase-3 activation, as well as stress features in mitochondria including ROS generation, compromised membrane integrity, altered respiration, and morphological changes. Moreover, PT-112 induced damage-associated molecular pattern (DAMP) release, the first demonstration of ICD in human cancer cell lines, in addition to autophagy initiation across the panel. Taken together, PT-112 caused selective stress, growth inhibition and death in human prostate cancer cell lines. Our data provide additional insight into mitochondrial stress and ICD in response to PT-112. PT-112 anticancer immunogenicity could have clinical applications and is currently under investigation in a Phase 2 mCRPC study.

Modulation of the Oncogenic LINE-1 Regulatory Network in Non-Small Cell Lung Cancer by Exosomal miRNAs.

Several microRNAs (miRNAs), including miR-221-5p, Let-7b-5p, miR-21-5p, miR-9-5p, miR-126-3p, and miR-222-3p, were recently found to be enriched in circulating exosomes of patients with non-small cell lung cancers (NSCLCs). These miRNAs distinguished cancer cases from controls with high precision and were predicted to modulate the expression of genes within the oncogenic LINE-1 regulatory network. To test this hypothesis, plasma exosomes from controls, early, and late-stage NSCLC patients were co-cultured with non-tumorigenic lung epithelial cells for 72 h and processed for measurements of gene expression. Exosomes from late-stage NSCLC patients markedly increased the mRNA levels of LINE-1 ORF1 and ORF2, as well as the levels of target miRNAs in naïve recipient cells compared to saline or control exosomes. Late-stage exosomes also modulated the expression of oncogenic targets within the LINE-1 regulatory network, namely, ICAM1, AGL, RGS3, RGS13, VCAM1, and TGFβ1. In sharp contrast, exosomes from controls or early-stage NSCLC patients inhibited LINE-1 expression, along with many of the genetic targets within the LINE-1 regulatory network. Thus, late-stage NSCLC exosomes activate LINE-1 and miRNA-regulated oncogenic signaling in non-tumorigenic, recipient lung bronchial epithelial cells. These findings raise important questions regarding lung cancer progression and metastasis and open the door for the exploration of new therapeutic interventions.

Alpha-1-antitrypsin improves anastomotic healing in intestinal epithelial cells model.

Intestinal anastomosis is a routine procedure in pediatric surgery, with leakage being a significantcomplication. Human alpha1-antitrypsin (AAT), whose physiological serum concentrations range from 0.9-2.0 mg/ml,is known to accelerate wound healing and stimulate the expression of cell proliferation-related genes. Wehypothesized that AAT might enhance anastomotic healing. In a monolayer of non-tumorigenic HIEC-6 epithelial cells derived from fetal intestine a scratch wascreated. Standard medium without (control) or with AAT (0.5 and 1 mg/ml) was added. Cells were observed using aLife-Cell Imaging System. Cell proliferation was assessed, and the expression of proliferation-related genes wasmeasured by qRT-PCR. In the presence of AAT, the scratch closed significantly faster. Cells treated with 1 mg/ml AAT showed 53%repopulation after 8 h and 97% after 18 h, while control cells showed 24% and 60% repopulation, respectively(p < 0.02). The treatment with AAT induced HIEC-6-cell proliferation and significantly increased the mRNA-expressionof CDKN1A, CDKN2A, ANGPTL4, WNT3 and COL3A1 genes. AAT did not change the mRNA-expression of CXCL8 butdecreased levels of IL-8 as compared to controls. At physiological concentrations AAT accelerates the confluence of intestinal cells and increases cellproliferation. The local administration of AAT may bear therapeutic potential to improve anastomotic healing.

A Novel HER2 Protein Identification Methodology in Breast Cancer Cells Using Raman Spectroscopy and Raman Imaging: An Analytical Validation Study.

Conventional assays such as immunohistochemistry (IHC) and in situ hybridization (ISH) used in clinical procedures for quantification of the human epidermal growth factor receptor-2 (HER2) status in breast cancer have many limitations. In the current study, we have used HER2 expression in a broad range of breast cancer phenotypes to explore the potential utility of a novel immunodetection technique using Raman spectroscopy and Raman imaging combined with artificial intelligence models. The correlations between the Raman method and conventional HER2 testing methodologies (IHC and ISH) have been tested. Raman measurements showed a strong linear correlation (p = 0.05, R2=0,9816) with IHC analysis in the studied breast cell lines: MCF-10A, MCF-7, MDA-MB-231, HTB-30 (SK-BR-3), and AU-565 represent normal, nontumorigenic epithelial cells, triple-positive breast carcinoma, and triple-negative breast cancer cell lines. Analytic testing of Raman spectroscopy and Raman imaging demonstrated that this method may offer advantages over the currently used diagnostic methodologies.

Gelatin nanoparticles loaded with 3-alkylpyridinium salt APS7, an analog of marine toxin, are a promising support in human lung cancer therapy

This study demonstrates the potential of gelatin nanoparticles as a nanodelivery system for antagonists of nicotinic acetylcholine receptors (nAChRs) to improve chemotherapy efficacy and reduce off-target effects. Too often, chemotherapy for lung cancer does not lead to satisfactory results. Therefore, new approaches directed at multiple pharmacological targets in cancer therapy are being developed. Following the activation of nAChRs (e.g. by nicotine), cancer cells begin to proliferate and become more resistant to chemotherapy-induced apoptosis. This work shows that the 3-alkylpyridinium salt, APS7, a synthetic analog of a toxin from the marine sponge Haliclona (Rhizoneira) sarai, acts as an nAChR antagonist that inhibits the pro-proliferative and anti-apoptotic effects of nicotine on A549 human lung adenocarcinoma cells. In this study, gelatin-based nanoparticles filled with APS7 (APS7-GNPs) were prepared and their effects on A549 cells were compared with that of free APS7. Both APS7 and APS7-GNPs inhibited Ca2+ influx and increased the efficacy of cisplatin chemotherapy in nicotine-stimulated A549 cells. However, significant benefits from APS7-GNPs were observed – a stronger reduction in the proliferation of A549 lung cancer cells and a much higher selectivity in cytotoxicity towards cancer cells compared with non-tumorigenic lung epithelial BEAS-2B cells.

Green synthesis of bimetallic AgZnO nanoparticles: Synergistic anticancer effects through regulation of gene expression for lung cancer treatment

Lung cancer is the foremost diagnosed cancer in men and the second most common cancer worldwide, highlighting the need for new treatment strategies. In the current study, AgZnO bimetallic nanoparticles (NPs) with average size of 11 nm were successfully synthesized using Salvia officinalis (SO) extract. The as-synthesized AgZnO NPs were characterized by several microscopic and spectroscopic techniques. The distribution of AgZnO nanoparticle curve was ranged from 10 to 20 nm, and the average particle diameter size was approximately 15.6 ± 4.3 nm with zeta potential value of −31.1 ± 5.2 mV. Also, the EDX technique revealed that the weight percentage for Zn, O, and Ag was 5.9 %, 3.6 %, and 90.5 %, respectively. The cytotoxicity of AgZnO NPs was assessed against A549 lung cancer cells and human non-tumorigenic lung epithelial BEAS-2B cells. The as-synthesized AgZnO NPs showed biocompatibility with heathy cells, while they showed high cytotoxicity against A549 lung cancer cells. In addition, AgZnO NPs demonstrated higher cytotoxicity compared to AgNPs synthesized by SO extract, indicating the synergistic effect between Ag and ZnO in the structure of bimetallic nanoparticle. The percentage of apoptosis and necrosis for SO extract was 11.6 % (3.69 % early apoptosis and 8.07 % late apoptosis) and 2.79 %, respectively. On the other hand, the apoptosis and necrosis percentage for AgZnO NPs was 29.74 % (5.04 % early apoptosis and 24.7 % late apoptosis) and 9.09 %, respectively. The high cytotoxicity effect was found through apoptosis, owing to upregulation of proapoptotic genes (caspase-3, caspase-8, caspase-9, P21, and BAX) and downregulation of antiapoptotic and migration genes (BCL2, cyclin E, cyclin D1, MMP-2, and MMP-9) in A549 cells. In addition, the A549 cells treated with AgZnO NPs illustrated lower migration ability compared to those treated with S. officinalis extract, indicating the role of AgZnO NP in cytotoxicity. Also, arrested cells during sub-G1 phase increased significantly after treatment with AgZnO NPs compared to SO extract (p < 0.05) (17.8 % for SO extract and 38 % for AgZnO NPs). The results of this study prove the enhanced anticancer effect of AgZnO NPs due to the synergistic effect between Ag and ZnO against the A549 lung cancer cells, which could be further investigated for cancer treatment application.

Cytotoxic Activity of the Red Grape Polyphenol Resveratrol against Human Prostate Cancer Cells: A Molecular Mechanism Mediated by Mobilization of Nuclear Copper and Generation of Reactive Oxygen Species.

Resveratrol, a polyphenolic compound found primarily in red grapes and pomegranates is known as an antioxidant but can act as a pro-oxidant when copper ions are present. Here, resveratrol is demonstrated to reduce cell growth (as evaluated by MTT assay) and promote apoptosis-like cell death (as measured by Histone/DNA ELISA) in prostate cancer cell lines PC3 and C42B. This effect is effectively inhibited by a copper chelator (neocuproine) and reactive oxygen species (ROS) scavengers (thiourea for hydroxyl radical, superoxide dismutase for superoxide anion, and catalase for hydrogen peroxide). These inhibitory effects provide evidence that intracellular copper reacts with resveratrol within cancer cells, resulting in DNA damage via the generation of reactive oxygen species. Additionally, it has been demonstrated that non-tumorigenic epithelial cell lines (MCF-10A) grown in media supplemented with copper are more susceptible to growth inhibition by resveratrol, as confirmed by the observed reduction in cell proliferation. Copper supplementation induces enhanced expression of the copper transporter CTR1 in MCF-10A cells, which is reduced by the addition of resveratrol to the media. The selective cell death of cancer cells generated by copper-mediated and ROS mechanisms may help to explain the anticancer properties of resveratrol.

Abstract PO3-25-03: Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there has been no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a expression in breast cancer associate with its aggressiveness and can be a prognostic biomarker. Material and Methods: Non-tumorigenic epithelial cell line, MCF-10A and breast cancer cell lines, MCF-7, T47D, and MDA-MB231, were used to analyze the expression levels of miR-125a as well as cell viability. The data of 2042 patients were extracted from publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC), The Cancer Genome Atlas (TCGA), and GSE57897. These databases included clinicopathological and survival data as well as transcriptome data. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by xCell. Results: The expression levels of miR-125a were suppressed in breast cancer cell lines, MCF-7, T47D, and MDA-MB231compared with MCF-10A. This was concordant with human samples. The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts. These results were in agreement with the notion that it is a tumor suppressive miRNA (p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p&amp;lt; 0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. To our surprise, high miR-125a tumors demonstrated worse disease free survival (DFS), disease specific survival (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, miR-125a expression was associated with Nottingham histological grade only in ER+/HER2- among the subtypes (p&amp;lt; 0.001). Also, high miR-125a tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- (p=0.02), but not in other subtypes. Furthermore, high miR-125a tumors enriched four out of five cell proliferation-related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to high miR-125a tumors in ER+/HER2-, but not in TNBC. Anti-tumor immune cells, CD8 cells, T-helper type 1 cells, and M1 macrophages, were all elevated in high miR-125a tumors in ER+/HER2- subtype (all p&amp;lt; 0.03), but none in TNBC. Also, pro-cancerous immune cell such as T-helper type 2 cells were similarly highly infiltrated with high miR-125a tumors only in ER+/HER2- subtype (p=0.026). Given its association with the survival outcome, we cannot help but speculate that miR-125a expression is parallel with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. Conclusion: High miR-125a levels in tumors were associated with aggressive biology and worse survival, thus it can be a candidate for a prognostic biomarker in ER+/HER2- patients. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mai Okawa, Saki Ando, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-03.

Modulation of the Effect of Cisplatin on Nicotine-Stimulated A549 Lung Cancer Cells Using Analog of Marine Sponge Toxin Loaded in Gelatin Nanoparticles.

Nicotine activates nicotinic acetylcholine receptors (nAChRs), which are overexpressed in numerous cancer types, leading to signaling pathways that increase lung cancer invasiveness and resistance to chemotherapeutic agents. In this study, the effects of APS12-2, a synthetic analog of marine sponge toxin that acts as an antagonist of nAChRs, was investigated in vitro on A549 human lung adenocarcinoma cells and non-tumorigenic human lung epithelial BEAS-2B cells. In addition, gelatin nanoparticles (GNPs) loaded with APS12-2 (APS12-2-GNPs) were prepared and their effects were compared with those of free APS12-2. Nicotine reduced cytotoxicity, the formation of reactive oxygen species, and the formation of lipid droplets caused by cisplatin on A549 cells. The effects of nicotine on the decreased efficacy of cisplatin were reduced by APS12-2 and APS12-2-GNPs. APS12-2-GNPs showed a substantial advantage compared with free APS12-2; the cytotoxicity of APS12-2 on BEAS-2B cells was greatly reduced when APS12-2 was loaded in GNPs, whereas the cytotoxicity on A549 cells was only slightly reduced. Our results suggest that both APS12-2 and APS12-2-GNPs hold promise as supportive agents in the cisplatin-based chemotherapy of lung cancer.

Reliability aspects in repulsive steric hindrance approach: Selectivity and sensitivity investigations

Dual metal regulated bottom gate top contact structured Organic Thin Film Transistor based biosensor (Bio-OTFT) for recognition of breast cancer cell lines along with the non-tumorigenic epithelial cell line is presented here. A feasible fabrication scheme for Bio-OTFT has been demonstrated. Repulsive steric hindrance (RSH) consideration inside the cavity zone directs more practical and realistic sensing mechanisms. The insightful electrostatic analysis regarding electric field, energy band diagram, transfer characteristics (IDS-VGS), and transconductance (gm) has been described. Alteration of the dielectric constant (k) of these cancerous and healthy cell lines stated the recognition scheme in this reported Bio-OTFT. The efficiency of the recommended model has been assessed thoroughly with the reported OTFT literatures. The sensitivity is examined respecting drain current (IDS) in (A/μm), gm in (μS/μm), threshold voltage (VTH) in Voltage and sub-threshold swing (SS) in (mV/dec) and reaches to the maximum value of 2.3 × 108, 1.85 × 1010, 0.68, and 0.08 for T-47D cell line at VGS = −1.25 V. The recommended model thus suggests an enormous reliability and possibility for biosensing purposes.

Abstract 4349: Characterization of a centrosome loss-induced tumorigenic signature in prostate epithelial cells

Abstract Prostate cancer (PCa) stands as the second leading cause of cancer death in American men, with overtreatment being common given the difficulty in distinguishing between indolent and aggressive cases. Unlike many cancers, PCa lacks hallmark mutations in key oncogenes and tumor-suppressor genes. Instead, PCa exhibit extensive genomic rearrangements and chromosomal instability (CIN). CIN occurs during PCa progression, producing ETS gene family fusions, PTEN loss and androgen receptor amplification. Importantly, the mechanism underlying CIN in PCa remains unclear. Previously, we found that cell within early-grade human primary prostate adenocarcinomas (PRAD) frequently lack centrosomes, and this frequency correlates with tumor grade. We demonstrated that transient removal of centrosomes within non-tumorigenic human prostate epithelial cells (hPrEC) induces CIN and was, strikingly, sufficient to transform subpopulations of cells capable of producing xenograft tumors in mice. To unravel the molecular mechanisms underlying this path to tumorigenesis, we isolated DNA and RNA from parental hPrECs, clonal lines subjected to transient centrosome loss, and xenograft tumor cells and performed whole genome sequencing (WGS) and bulk RNA-seq. This allowed us to characterize the genomic profiles induced by centrosome loss and to identify an associated mutational signature. We used MUTECT2 to identify single nucleotide variants. Kataegis loci were observed on multiple chromosomes in all transient centrosome removal samples. Copy number variations were also detected in these samples using FACETS and SEQUENZA. Next, we extracted the copy number (CN) signature with SigProfiler and Sigminer, comparing it to COSMIC CN signatures and WGS data from PRAD patients. Our results revealed that the transient centrosome loss signature bears similarity to CN signatures associated with chromothripsis, loss of heterozygosity, and homologous recombination repair deficiency. Patients with PRAD displaying the centrosome loss CN signature had a poorer prognosis. Additionally, our CN analysis discovered that centrosome loss induced mosaic loss of chromosome Y in our samples. To detect structural variations, we employed DELLY, MANTA, SVABA, revealing a high occurrence of both non-clustered and clustered translocations in transient centrosome loss samples. These translocations were validated in our RNA-seq data using STAR-Fusion. Ongoing analyses include inferring CNV from PCa scRNA-seq data, and determining cell populations with the centrosome loss CN-signature and their associated transcriptome features. This work unveils a comprehensive genomic profile stemming from centrosome loss and demonstrates its role in driving oncogenesis in PCa. These findings have the potential to establish centrosome loss as a hallmark in PCa stratification, and lead to a significant advancement in prostate tumor treatment. Citation Format: Jiawen Yang, Diogo de Oliveira Pessoa, John M. Ryniawec, Emily Loertscher, Anne E. Cress, Megha Padi, Gregory C. Rogers. Characterization of a centrosome loss-induced tumorigenic signature in prostate epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4349.

Abstract 1881: Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds

Abstract Metastasis is responsible for &amp;gt;90% of cancer-related deaths and is a key cause of failure of cancer therapy. The development of effective targeted therapy to prevent and treat metastasis has been challenging. Homologous Rho GTPases Rac and Cdc42 are viable metastasis targets because they have a crucial role in the signaling pathways that regulate cell survival, proliferation, and migration, so dysregulation of these proteins leads to cancer progression and metastasis of diverse cancer types. Therefore, targeting these regulatory proteins results in anticancer and antimetastatic activity, as observed with our dual Rac/Cdc42 inhibitor, MBQ-167, in triple-negative breast cancer. MBQ-167 has an IC50 of ~100nM for Rac and Cdc42 activation inhibition and a GI50 of 130nM. MBQ-167 inhibits the binding of guanine nucleotides to Rac and Cdc42 with similar efficacy. The purpose of this study was to identify MBQ-167 derivatives with similar or greater efficacy and specificity, with a range of inhibitory mechanisms, that may be specific to the different guanine nucleotide exchange factors expressed in distinct cancer cell types. Currently, over 30 different compounds with potential Rac1 and Cdc42 inhibitory activity are being evaluated. So far, some of the evaluated compounds have shown comparable GI50 values to MBQ-167 in metastatic breast cancer cell lines. From this screening, CPV-337 was selected as a compound with a GI50 of ~55nM and ~70% Rac activation inhibition at 50nM, therefore being 2X more effective than our leading compound MBQ-167. However, evaluation of CPV-337 in the viability of the MCF-10 non-tumorigenic epithelial cell line demonstrated a GI50 of ~63nM, which is similar to that of breast cancer cells. Therefore, CPV-337 may have cytotoxic effects on breast cancer. We are continuing to screen the library of MBQ-167 derivatives in breast, pancreatic, lung, and prostate cancer cell lines. Citation Format: Jessica Colón González, María C. Santa María Fuentes, Cornelis Vlaar, Suranganie Dharmawardhane. Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1881.

Ionic Liquid-Based Polymer Matrices for Single and Dual Drug Delivery: Impact of Structural Topology on Characteristics and In Vitro Delivery Efficiency.

Previously reported amphiphilic linear and graft copolymers, derived from the ionic liquid [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TMAMA_Cl‾), along with their conjugates obtained through modification either before or after polymerization with p-aminosalicylate anions (TMAMA_PAS‾), were employed as matrices in drug delivery systems (DDSs). Based on the counterion type in TMAMA units, they were categorized into single drug systems, manifesting as ionic polymers with chloride counterions and loaded isoniazid (ISO), and dual drug systems, featuring ISO loaded in self-assembled PAS conjugates. The amphiphilic nature of these copolymers was substantiated through the determination of the critical micelle concentration (CMC), revealing an increase in values post-ion exchange (from 0.011-0.063 mg/mL to 0.027-0.181 mg/mL). The self-assembling properties were favorable for ISO encapsulation, with drug loading content (DLC) ranging between 15 and 85% in both single and dual systems. In vitro studies indicated ISO release percentages between 16 and 61% and PAS release percentages between 20 and 98%. Basic cytotoxicity assessments using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) test affirmed the non-toxicity of the studied systems toward human non-tumorigenic lung epithelial cell line (BEAS-2B) cell lines, particularly in the case of dual systems bearing both ISO and PAS simultaneously. These results confirmed the effectiveness of polymeric carriers in drug delivery, demonstrating their potential for co-delivery in combination therapy.

In Vitro and In Silico Studies on 4-Nitroacetophenone Thiosemicarbazone Potential Cytotoxicity Against A549 Cell Lines.

Lung malignancy is a major worldwide issue that occurs due to the dysregulation of various growth factors. Lung cancer has no apparent signs in the early stages, which makes it harder to catch it in time and leads to a higher fatality rate. So, the goal of this work was to create and analyze a novel chemical molecule called 4-nitro acetophenone thiosemicarbazone (4-NAPTSc) against the lung cancer cell line A549 and human non-tumorigenic lung epithelial cell line BAES-2B. The ligand was synthesized by refluxing the reaction mixture of 4-nitro acetophenone and thiosemicarbazide and was further characterized by UV, FTIR, and 1H and 13C NMR and Differential Scanning Calorimetry (DSC) study. Cytotoxicity assay/MTT (3-(4,5-dimethylthiazol-2-yl))2,5-diphenyltetrazolium bromide) was used to evaluate the cytotoxicity of the compound. Epidermal growth factor receptors (EGFR), polo-like kinase-1 (PLK1), and vascular endothelial growth factor receptors (VEGFR) were chosen as the target proteins for molecular docking to find potential ligand binding sites and inhibit their function. A novel yellow-colored crystalline solid has been synthesized. 4-NAPTSc had an IC50 of 2.93 μg/mL against the A549 lung cancer cells. When the dosage is increased from 5 to 15 μg/mL along with time, the cell viability falls. Docking results showed that the compound binds with the targeted proteins' amino acid residues, and the likeness profile of the compound is also favorable. This study reveals that the compound has the potential for further investigation and can be used in multitargeted cancer therapies.

In vitro and in vivo anticancer activity of mebendazole in colon cancer: a promising drug repositioning.

Colon cancer is one of the most common cancers and one of the main causes of death worldwide. Therefore, new treatment methods with better efficiency and fewer risks are very necessary. Mebendazole (MBZ), a drug commonly used for helminthic infections, has recently received attention as a suitable candidate for the treatment of various cancers. This study aimed to investigate, in vitro and in vivo, anticancer activity and selectivity Index of MBZ on colon cancer. HT-29 (human colorectal adenocarcinoma) and MCF-10 (non-tumorigenic epithelial) cell lines were treated with MBZ and Doxorubicin (DOX; positive control drug). IC50 values were estimated using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay. We employed flow cytometry using annexin V-FITC and propidium iodide dyes. For the animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in Bulb/C mice. The mice were treated with 0.05 of LD50, intraperitoneal, every other day for 35 days. Finally, the survival rate, tumor volume, and tumor weight were calculated. Our results demonstrated that IC50 values after 72 h for HT29 and MCF-10 cell lines were 0.29 ± 0.04 µM and 0.80 ± 0.02 µM, respectively. MBZ was more selective than DOX in inhibiting the proliferation of cancer cells compared to normal cells (2. 75 vs. 2.45). Annexin V/PI staining demonstrated that MBZ treatment at IC50 concentrations induced (78 ± 12%) apoptosis in the HT29 cancer cell line after 48 h (P ≤ 0.0001). Also, in mice bearing colon cancer, MBZ significantly reduced the tumor volume (1177 ± 1109 mm3; P ≤ 0.001) and tumor weight (2.30 ± 1.97 g; P ≤ 0.0001) compared to the negative control group (weight 12.45 ± 2.0 g; volume 7346 ± 1077). Also, MBZ increases mean survival time (MST) and increase life span (ILS) percentage in the animal study (51.2 ± 37% vs 93%, respectively). This study suggests that mebendazole strongly and selectively inhibits proliferation and induces apoptosis in colon cancer cells. It may be, accordingly, a promising drug for clinical research and application.

Hydrogen Peroxide Mediates Pharmacological Ascorbate Induced Radio-Sensitization of Pancreatic Cancer Cells by Enhancing G2-accumulation and Reducing Cyclin B1 Protein Levels.

Pharmacological ascorbate (P-AscH-, high dose, intravenous vitamin C) preferentially sensitizes human pancreas ductal adenocarcinoma (PDAC) cells to radiation-induced toxicity compared to non-tumorigenic epithelial cells. Radiation-induced G2-checkpoint activation contributes to the resistance of cancer cells to DNA damage induced toxicity. We hypothesized that P-AscH- induced radio-sensitization of PDAC cells is mediated by perturbations in the radiation induced activation of the G2-checkpoint pathway. Both non-tumorigenic pancreatic ductal epithelial and PDAC cells display decreased clonogenic survival and increased doubling times after radiation treatment. In contrast, the addition of P-AscH- to radiation increases clonogenic survival and decreases the doubling time of non-tumorigenic epithelial cells but decreasing clonogenic survival and increasing the doubling time of PDAC cells. Results from the mitotic index and propidium iodide assays showed that while the P-AscH- treatments did not affect radiation-induced G2-checkpoint activation, it enhanced G2-accumulation. The addition of catalase reverses the increases in G2-accumulation, indicating a peroxide-mediated mechanism. In addition, P-AscH- treatment of PDAC cells suppresses radiation-induced accumulation of cyclin B1 protein levels. Both translational and post-translational pathways appear to regulate cyclin B1 protein levels after the combination treatment of PDAC cells with P-AscH- and radiation. The protein changes seen are reversed by the addition of catalase suggesting that hydrogen peroxide mediates P-AscH- induced radiation sensitization of PDAC cells by enhancing G2-accumulation and reducing cyclin B1 protein levels.

Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor.

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 μM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 μM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

The Inhibition of Human Epithelial Cells Proliferation by Oridoninthrough Interrupted Notch Signalling Pathway

Since the Notch signaling pathway plays a vital role in cell proliferation, oridonin restrains cancer cell growth bylimiting the Notch pathway. The study aims to investigate whether oridonin can kill normal epithelial cells similar tocancer cells in vitro. Also, the study aims to find whether the inhibition of Notch-1 by oridonin is on a transcriptionallevel. Methods: The study will use human epithelial cells (MCF10A) and breast cancer cells (MCF7). These two typesof cells will be treated with various concentrations of oridonin (0.01mM, 0.1mM, 1mM, and 10mM) for a differentamount of time (24h, 48h, 72h). The cell viability will be measured by WST-1 assay. Possible Results: There areeight possible results, three for the proliferation of epithelial cells and two for the transcriptional level of Notch-1 inepithelial cells. Normal epithelial cells experience a more significant loss of viable cells compared with cancer cells:(1) with decreased transcription and expression of Notch-1; (2) with decreased expression of Notch-1 but no decreasein transcription; (3) with a decrease in transcription but no decrease in expression of Notch-1 (4) with no decreasein expression and transcription of Notch-1. Normal epithelial cells experience a similar or less loss of viable cellscompared with cancer cells, (5) with decreased transcription and expression of Notch-1; (6) with decreased expressionof Notch-1 but no decrease in transcription; (7) with a decrease in transcription but no decrease in expression of Notch-1(8) with no decrease in expression and transcription of Notch-1. Conclusion: The loss of normal cells is considered atrade-off for the anti-tumor activity of oridonin. If little or no damage to non-tumorigenic epithelial cells is detected,then oridonin may be a good cure for breast cancer with fewer side effects. Also, by investigating whether the inhibitionof Notch-1 by oridonin is on a transcriptional level, we can gain more insights into the mechanism of inhibition.

ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7.

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). Thus far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether the upregulation of candidate polyamine transporters of the P5B transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells presented elevated expression of a previously uncharacterized P5B-ATPase, ATP13A4, which was responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells were more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, the overexpression of ATP13A4 WT in MCF10A cells induced a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labeled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we established ATP13A4 as a new polyamine transporter in the human PTS and showed that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.