In Vitro and In Silico Biological Studies of 4-Phenyl-2-quinolone (4-PQ) Derivatives as Anticancer Agents.

Our previous study found that 2-phenyl-4-quinolone (2-PQ) derivatives are antimitotic agents, and we adopted the drug design concept of scaffold hopping to replace the 2-aromatic ring of 2-PQs with a 4-aromatic ring, representing 4-phenyl-2-quinolones (4-PQs). The 4-PQ compounds, whose structural backbones also mimic analogs of podophyllotoxin (PPT), maybe a new class of anticancer drugs with simplified PPT structures. In addition, 4-PQs are a new generation of anticancer lead compounds as apoptosis stimulators. On the other hand, previous studies showed that 4-arylcoumarin derivatives with 5-, 6-, and 7-methoxy substitutions displayed remarkable anticancer activities. Therefore, we further synthesized a series of 5-, 6-, and 7-methoxy-substituted 4-PQ derivatives (19-32) by Knorr quinoline cyclization, and examined their anticancer effectiveness. Among these 4-PQs, compound 22 demonstrated excellent antiproliferative activities against the COLO205 cell line (50% inhibitory concentration (IC50) = 0.32 µM) and H460 cell line (IC50 = 0.89 µM). Furthermore, we utilized molecular docking studies to explain the possible anticancer mechanisms of these 4-PQs by the docking mode in the colchicine-binding pocket of the tubulin receptor. Consequently, we selected the candidate compounds 19, 20, 21, 22, 25, 27, and 28 to predict their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles. Pharmacokinetics (PKs) indicated that these 4-PQs displayed good drug-likeness and bioavailability, and had no cardiotoxic side effects or carcinogenicity, but we detected risks of drug-drug interactions and AMES toxicity (mutagenic). However, structural modifications of these 4-PQs could improve their PK properties and reduce their side effects, and their promising anticancer activities attracted our attention for further studies.

Deciphering the immuno-pathological role of FLT, and evaluation of a novel dual inhibitor of topoisomerases and mutant-FLT3 for treating leukemia.

Acute myeloid leukemia (AML) is a type of leukemia with an aggressive phenotype, that commonly occurs in adults and with disappointing treatment outcomes. Genetic alterations were implicated in the etiology of cancers and form the basis for defining patient prognoses and guiding targeted therapies. In the present study, we leveraged bulk and single-cell RNA sequencing datasets from AML patients to determine the clinical significance of Fms-related receptor tyrosine kinase 3 (FLT3) alterations on the T-cell phenotype and immune response of AML patients. Subsequently, we evaluated the therapeutic potential of Lwk-n019, a novel small-molecule derivative of thiochromeno[2,3-c]quinolin-12-one. Our results suggested that FLT3 plays an important role in the progression, aggressive phenotype, and worse immune response of patients. An FLT3 mutation was associated with dysfunctional T-cell phenotypes, and high risk and shorter survival of AML patients. Our findings further suggested that the aggressiveness of AML and the prognostic role of FLT3 are associated with the co-occurrence of NPM1 and DNMT3A mutations. Lwk-n019 demonstrated dose-dependent anticancer activities against various leukemia cancer cell lines. Lwk-n019 demonstrated highly selective kinase inhibitory activities against the wild-type FLT3 (D835V) and mutant FLT3 (internal tandem duplication (ITD), D835V) with >95% and 99% inhibitory levels, respectively. Moreover, the compound demonstrated the best binding constant (Kd value) of 0.77 µM against FLT3 (ITD, 835V). In addition, Lwk-n019 significantly inhibited the activities of both the topoisomerase I (TOPI) and TOPII enzymes, with higher TOPI inhibitory activity than camptothecin, a clinical inhibitor. While the jejunum, duodenum, cecum, and colon were prime sites of absorption, Lwk-n019 achieved maximum concentration (Cmax), Vd, blood/plasma ratio, time to maximum concentration (Tmax), area under the receiver operating concentration curve (AUC)(0-24), and AUC(0-∞) values of 0.665 µg/mL, 5.21 Vc, L/kg, 1.5 h, 6634.7, and 6909.2, respectively. In conclusion, Lwk-n019 demonstrated anticancer activities via multi-target inhibition of TOPs and kinases with high inhibition preference for mutant ITD-FLT3. The present pioneer study provides a basis for advanced optimization of drug potency, selectivity, specificity, and other properties desired of anticancer drug leads. Studies are ongoing to determine the full therapeutic properties of Lwk-n019 and the detailed mechanisms of FLT3 in TOP inhibition.

Erratum: A preclinical report of a cobimetinib-inspired novel anticancer small-molecule scaffold of isoflavones, NSC777213, for targeting PI3K/AKT/mTOR/MEK in multiple cancers.

[This corrects the article on p. 2590 in vol. 11, PMID: 34249417.].

In vivo Pharmacokinetic and Anticancer Studies of HH-N25, a Selective Inhibitor of Topoisomerase I, and Hormonal Signaling for Treating Breast Cancer.

PURPOSE: Breast cancer is the most frequently diagnosed cancer globally, and the leading cause of cancer-associated mortality among women. The efficacy of most clinical chemotherapies is often limited by poor pharmacokinetics and the development of drug resistance by tumors. In a continuing effort to explore small molecules as alternative therapies, we herein evaluated the therapeutic potential of HH-N25, a novel nitrogen-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-dione derivative. METHODS: We evaluated the in vivo pharmacokinetic properties and maximum tolerated dose (MTD) of HH-N25 in rats. We also characterized the compound for in vitro and in vivo anticancer activities and its inhibitory effects against DNA topoisomerases and hormonal signaling in breast cancer. Furthermore, we used molecular docking to analyse the ligand-receptor interactions between the compound and the targets. RESULTS: The maximum serum concentration (Cmax), half-life (t1/2 beta), mean residence time (MRT), oral clearance (CL/f), and apparent volume of distribution (VD/f) of HH-N25 were 1446.67 ± 312.05 ng/mL, 4.51 ± 0.27 h, 2.56 ± 0.16 h, 8.32 ± 1.45 mL/kg/h, and 1.26 ± 0.15 mL/kg, respectively, after single-dose iv administration at 3 mg/kg body weight. HH-N25 had potent anticancer activity against a panel of human breast cancer cell lines with 50% inhibitory concentrations (IC50) ranging 0.045±0.01~4.21±0.05 µM. The drug also demonstrated marked in vivo anticancer activity at a tolerated dose and prolonged the survival duration of mice without unacceptable toxicities based on body weight changes in human tumor xenograft models. In addition, HH-N25 exhibited a dose-dependent inhibition of topoisomerase I and ligand-mediated activities of progesterone and androgen receptors. CONCLUSION: HH-N25 represents a new molecular entity that selective suppressed TOP1 and hormonal signaling, and shows potent antitumor activities in human breast cancer cells in vitro and in vivo. HH-N25 thus represents a promising anticancer agent that warrants further preclinical and clinical exploration.

LCC-09, a Novel Salicylanilide Derivative, Exerts Anti-Inflammatory Effect in Vascular Endothelial Cells.

OBJECTIVE: Endothelial cell (EC) activation facilitates leukocyte adhesion to vascular walls, which is implicated in a variety of cardiovascular diseases and is a target for prevention and treatment. Despite the development of anti-inflammatory medications, cost-effective therapies with significant anti-inflammatory effects and lower organ toxicity remain elusive. The goal of this study is to identify novel synthetic compounds that inhibit EC inflammatory response with minimal organ toxicity. METHODS AND RESULTS: In this study, we discovered LCC-09, a salicylanilide derivative consisting of the functional fragment of magnolol, 2,4-difluorophenyl, and paeonol moiety of salicylate, as a novel anti-inflammatory compound in cultured ECs and zebrafish model. LCC-09 was shown to inhibit pro-inflammatory cytokine tumor necrosis factor-α (TNFα)-induced expression of adhesion molecules and inflammatory cytokines, leading to reduced leukocyte adhesion to ECs. Mechanistically, LCC-09 inhibits the phosphorylation of signal transducer and activator of transcription 1 (STAT1), TNFα-induced degradation of NF-κ-B Inhibitor-α (IκBα) and phosphorylation of NFκB p65, resulting in reduced NFκB transactivation activity and binding to E-selectin promoter. Additionally, LCC-09 attenuated TNFα-induced generation of reactive oxygen species in ECs. Molecular docking models suggest the binding of LCC-09 to NFκB essential modulator (NEMO) and Janus tyrosine kinase (JAK) may lead to dual inhibition of NFκB and STAT1. Furthermore, the anti-inflammatory effect of LCC-09 was validated in the lipopolysaccharides (LPS)-induced inflammation model in zebrafish. Our results demonstrated that LCC-09 significantly reduced the LPS-induced leukocyte recruitment and mortality of zebrafish embryos. Finally, LCC-09 was administered to cultured ECs and zebrafish embryos and showed minimal toxicities. CONCLUSION: Our results support that LCC-09 inhibits EC inflammatory response but does not elicit significant toxicity.

A preclinical report of a cobimetinib-inspired novel anticancer small-molecule scaffold of isoflavones, NSC777213, for targeting PI3K/AKT/mTOR/MEK in multiple cancers.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways are critical for normal human physiology, and any alteration in their regulation leads to several human cancers. These pathways are well interconnected and share a survival mechanism for escaping the depressant effect of antagonists. Therefore, novel small molecules capable of targeting both pathways with minimal or no toxicity are better alternatives to current drugs, which are disadvantaged by their accompanying resistance and toxicity. In this study, we demonstrate that the PI3K/AKT/mTOR/MEK is a crucial oncoimmune signature in multiple cancers. Moreover, we describe NSC777213, a novel isoflavone core and cobimetinib-inspired small molecule, which exhibit both antiproliferative activities against all panels of NCI60 human tumor cell lines (except COLO205 and HT29) and a selective cytotoxic preference for melanoma, non-small-cell lung cancer (NSCLC), brain, renal, and ovarian cancer cell lines. Notably, for NSC777213 treatment, chemoresistant ovarian cancer cell lines, including SK-OV-3, OVCAR-3, OVCAR-4, and NCI/ADR-RES, exhibited a higher antiproliferative sensitivity (total growth inhibition (TGI) = 7.62-31.50 µM) than did the parental cell lines OVCAR-8 and IGROV1 (TGI > 100 µM). NSC777213 had a mechanistic correlation with clinical inhibitors of PI3K/AKT/mTOR/MEK. NSC777213 demonstrates robust binding interactions and higher affinities for AKT and mTOR than did isoflavone, and also demonstrate a higher affinity for human MEK-1 kinase than some MEK inhibitors under clinical developments. In addition, treatment of U251 and U87MG cells with NSC777213 significantly downregulated the expression levels of the total and phosphorylated forms of PI3K/AKT/mTOR/MEK. Our study suggests that NSC777213 is a promising PI3K/AKT/mTOR/MEK inhibitor for further preclinical and clinical evaluation as a chemotherapeutic agent, particularly for the treatment of NSCLC, melanoma, and brain, renal, and ovarian cancers.

Network Pharmacological Analysis through a Bioinformatics Approach of Novel NSC765600 and NSC765691 Compounds as Potential Inhibitors of CCND1/CDK4/PLK1/CD44 in Cancer Types.

Cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) both play significant roles in regulating cell cycle progression, while polo-like kinase 1 (PLK1) regulates cell differentiation and tumor progression, and activates cancer stem cells (CSCs), with the cluster of differentiation 44 (CD44) surface marker mostly being expressed. These oncogenes have emerged as promoters of metastasis in a variety of cancer types. In this study, we employed comprehensive computational and bioinformatics analyses to predict drug targets of our novel small molecules, NSC765600 and NSC765691, respectively derived from diflunisal and fostamatinib. The target prediction tools identified CCND1/CDK4/PLK1/CD44 as target genes for NSC765600 and NSC765691 compounds. Additionally, the results of our in silico molecular docking analysis showed unique ligand-protein interactions with putative binding affinities of NSC765600 and NSC765691 with CCND1/CDK4/PLK1/CD44 oncogenic signaling pathways. Moreover, we used drug-likeness precepts as our guidelines for drug design and development, and found that both compounds passed the drug-likeness criteria of molecular weight, polarity, solubility, saturation, flexibility, and lipophilicity, and also exhibited acceptable pharmacokinetic properties. Furthermore, we used development therapeutics program (DTP) algorithms and identified similar fingerprints and mechanisms of NSC765600 and NSC765691 with synthetic compounds and standard anticancer agents in the NCI database. We found that NSC765600 and NSC765691 displayed antiproliferative and cytotoxic effects against a panel of NCI-60 cancer cell lines. Based on these finding, NSC765600 and NSC765691 exhibited satisfactory levels of safety with regard to toxicity, and met all of the required criteria for drug-likeness precepts. Currently, further in vitro and in vivo investigations in tumor-bearing mice are in progress to study the potential treatment efficacies of the novel NSC765600 and NSC765691 small molecules.

In-Silico Evaluation of Genetic Alterations in Ovarian Carcinoma and Therapeutic Efficacy of NSC777201, as a Novel Multi-Target Agent for TTK, NEK2, and CDK1.

Ovarian cancer is often detected at the advanced stages at the time of initial diagnosis. Early-stage diagnosis is difficult due to its asymptomatic nature, where less than 30% of 5-year survival has been noticed. The underlying molecular events associated with the disease's pathogenesis have yet to be fully elucidated. Thus, the identification of prognostic biomarkers as well as developing novel therapeutic agents for targeting these markers become relevant. Herein, we identified 264 differentially expressed genes (DEGs) common in four ovarian cancer datasets (GSE14407, GSE18520, GSE26712, GSE54388), respectively. We constructed a protein-protein interaction (PPI) interaction network with the overexpressed genes (72 genes) and performed gene enrichment analysis. In the PPI networks, three proteins; TTK Protein Kinase (TTK), NIMA Related Kinase 2 (NEK2), and cyclin-dependent kinase (CDK1) with higher node degrees were further evaluated as therapeutic targets for our novel multi-target small molecule NSC777201. We found that the upregulated DEGs were enriched in KEGG and gene ontologies associated with ovarian cancer progression, female gamete association, otic vesicle development, regulation of chromosome segregation, and therapeutic failure. In addition to the PPI network, ingenuity pathway analysis also implicate TTK, NEK2, and CDK1 in the elevated salvage pyrimidine and pyridoxal pathways in ovarian cancer. The TTK, NEK2, and CDK1 are over-expressed, demonstrating a high frequency of genetic alterations, and are associated with poor prognosis of ovarian cancer cohorts. Interestingly, NSC777201 demonstrated anti-proliferative and cytotoxic activities (GI50 = 1.6 µM~1.82 µM and TGI50 = 3.5 µM~3.63 µM) against the NCI panels of ovarian cancer cell lines and exhibited a robust interaction with stronger affinities for TTK, NEK2, and CDK1, than do the standard drug, paclitaxel. NSC777201 displayed desirable properties of a drug-like candidate and thus could be considered as a novel small molecule for treating ovarian carcinoma.

mTOR/EGFR/iNOS/MAP2K1/FGFR/TGFB1 Are Druggable Candidates for N-(2,4-Difluorophenyl)-2',4'-Difluoro-4-Hydroxybiphenyl-3-Carboxamide (NSC765598), With Consequent Anticancer Implications.

BACKGROUND: The application of computational and multi-omics approaches has aided our understanding of carcinogenesis and the development of therapeutic strategies. NSC765598 is a novel small molecule derivative of salicylanilide. This study aims to investigate the ligand-protein interactions of NSC765598 with its potential targets and to evaluate its anticancer activities in vitro. METHODS: We used multi-computational tools and clinical databases, respectively, to identify the potential drug target for NSC765598 and analyze the genetic profile and prognostic relevance of the targets in multiple cancers. We evaluated the in vitro anticancer activities against the National Cancer Institute 60 (NCI60) human tumor cell lines and used molecular docking to study the ligand-protein interactions. Finally, we used the DTP-COMPARE algorithm to compare the NSC765598 anticancer fingerprints with NCI standard agents. RESULTS: We identified mammalian target of rapamycin (mTOR)/epidermal growth factor receptor (EGFR)/inducible nitric oxide synthase (iNOS)/mitogen-activated protein 2 kinase 1 (MAP2K1)/fibroblast growth factor receptor (FGFR)/transforming growth factor-ß1 (TGFB1) as potential targets for NSC765598. The targets were enriched in cancer-associated pathways, were overexpressed and were of prognostic relevance in multiple cancers. Among the identified targets, genetic alterations occurred most frequently in EGFR (7%), particularly in glioblastoma, esophageal squamous cell cancer, head and neck squamous cell cancer, and non-small-cell lung cancer, and were associated with poor prognoses and survival of patients, while other targets were less frequently altered. NSC765598 displayed selective antiproliferative and cytotoxic preferences for NSCLC (50% growth inhibition (GI50) = 1.12-3.95 µM; total growth inhibition (TGI) = 3.72-16.60 µM), leukemia (GI50 = 1.20-3.10 µM; TGI = 3.90-12.70 µM), melanoma (GI50 = 1.45-3.59 µM), and renal cancer (GI50 = 1.38-3.40 µM; TGI = 4.84-13.70 µM) cell lines, while panels of colon, breast, ovarian, prostate, and central nervous system (CNS) cancer cell lines were less sensitive to NSC765598. Interestingly, NSC765598 docked well into the binding cavity of the targets by conventional H-bonds, van der Waal forces, and a variety of π-interactions, with higher preferences for EGFR (ΔG = -11.0 kcal/mol), NOS2 (ΔG = -11.0 kcal/mol), and mTOR (ΔG = -8.8 kcal/mol). NSC765598 shares similar anti-cancer fingerprints with NCI standard agents displayed acceptable physicochemical values and met the criteria of drug-likeness. CONCLUSION: NSC765598 displayed significant anticancer and potential multi-target properties, thus serve as a novel candidate worthy of further preclinical studies.

An Integrated Bioinformatics Study of a Novel Niclosamide Derivative, NSC765689, a Potential GSK3ß/ß-Catenin/STAT3/CD44 Suppressor with Anti-Glioblastoma Properties.

Despite management efforts with standard surgery, radiation, and chemotherapy, glioblastoma multiform (GBM) remains resistant to treatment, which leads to tumor recurrence due to glioma stem cells (GSCs) and therapy resistance. In this study, we used random computer-based prediction and target identification to assess activities of our newly synthesized niclosamide-derived compound, NSC765689, to target GBM oncogenic signaling. Using target prediction analyses, we identified glycogen synthase kinase 3ß (GSK3ß), ß-Catenin, signal transducer and activator of transcription 3 (STAT3), and cluster of differentiation 44 (CD44) as potential druggable candidates of NSC765689. The above-mentioned signaling pathways were also predicted to be overexpressed in GBM tumor samples compared to adjacent normal samples. In addition, using bioinformatics tools, we also identified microRNA (miR)-135b as one of the most suppressed microRNAs in GBM samples, which was reported to be upregulated through inhibition of GSK3ß, and subsequently suppresses GBM tumorigenic properties and stemness. We further performed in silico molecular docking of NSC765689 with GBM oncogenes; GSK3ß, ß-Catenin, and STAT3, and the stem cell marker, CD44, to predict protein-ligand interactions. The results indicated that NSC765689 exhibited stronger binding affinities compared to its predecessor, LCC09, which was recently published by our laboratory, and was proven to inhibit GBM stemness and resistance. Moreover, we used available US National Cancer Institute (NCI) 60 human tumor cell lines to screen in vitro anticancer effects, including the anti-proliferative and cytotoxic activities of NSC765689 against GBM cells, and 50% cell growth inhibition (GI50) values ranged 0.23~5.13 µM. In summary, using computer-based predictions and target identification revealed that NSC765689 may be a potential pharmacological lead compound which can regulate GBM oncogene (GSK3ß/ß-Catenin/STAT3/CD44) signaling and upregulate the miR-135b tumor suppressor. Therefore, further in vitro and in vivo investigations will be performed to validate the efficacy of NSC765689 as a novel potential GBM therapeutic.

Pharmacoinformatics and Preclinical Studies of NSC765690 and NSC765599, Potential STAT3/CDK2/4/6 Inhibitors with Antitumor Activities against NCI60 Human Tumor Cell Lines.

Signal transducer and activator of transcription 3 (STAT3) is a transcriptional regulator of a number of biological processes including cell differentiation, proliferation, survival, and angiogenesis, while cyclin-dependent kinases (CDKs) are a critical regulator of cell cycle progression. These proteins appear to play central roles in angiogenesis and cell survival and are widely implicated in tumor progression. In this study, we used the well-characterized US National Cancer Institute 60 (NCI60) human tumor cell lines to screen the in vitro anti-cancer activities of our novel small molecule derivatives (NSC765690 and NSC765599) of salicylanilide. Furthermore, we used the DTP-COMPARE algorithm and in silico drug target prediction to identify the potential molecular targets, and finally, we used molecular docking to assess the interaction between the compounds and prominent potential targets. We found that NSC765690 and NSC765599 exhibited an anti-proliferative effect against the 60 panels of NCI human cancer cell lines, and dose-dependent cytotoxic preference for NSCLC, melanoma, renal, and breast cancer cell lines. Protein-ligand interactions studies revealed that NSC765690 and NSC765599 were favored ligands for STAT3/CDK2/4/6. Moreover, cyclization of the salicylanilide core scaffold of NSC765690 mediated its higher anti-cancer activities and had greater potential to interact with STAT3/CDK2/4/6 than did NSC765599 with an open-ring structure. NSC765690 and NSC765599 met the required safety and criteria of a good drug candidate, and are thus worthy of further in-vitro and in-vivo investigations in tumor-bearing mice to assess their full therapeutic efficacy.

Preclinical investigation of ovatodiolide as a potential inhibitor of colon cancer stem cells via downregulating sphere-derived exosomal ß-catenin/STAT3/miR-1246 cargoes.

Patients with advanced-stage colon cancer often exhibit resistance against treatment and distant metastasis, both key contributors to poor prognosis. Emerging evidence indicates that cancer stem cells (CSCs), characterized by the enhanced ability to self-renew, resist therapeutics, and promote metastasis, represents a clinical challenge to target. Alternative therapeutic approaches are urgently required. Here, we explored the feasibility of disrupting the intracellular communications between CSCs and the tumor microenvironment by way of exosomes. First, we demonstrated that exosomes secreted by colon tumorspheres (Exosp) promoted 5-FU resistance, migration, and tumorsphere formation. Exosp also increased the generation of cancer-associated fibroblasts and M2 polarized macrophages in vitro. Oncogenic molecules, including IL-6, p-STAT3, TGF-ß1, and ß-catenin, were identified as the cargoes of Exosp. Furthermore, the public database revealed the high abundance of miR-1246 in serum exosomes from colon cancer patients, and we verified in the Exosp from HCT116 and HT29 cells. Therapeutically, we demonstrated the ovatodiolide treatment reduced exosomal cargoes from tumorspheres (Exosp_OV). Exosp_OV were significantly less capable of promoting 5-FU resistance, migration, and tumorsphere formation when co-cultured with HCT116 and HT29 cells. Notably, Exosp_OV was less CAF- and M2 TAM-transformative. Computational docking analysis revealed that OV could bind and significantly reduced ß-catenin activity. Finally, mouse xenograft data indicated that ovatodiolide suppressed tumor growth via down-regulating IL-6, STAT3, ß-catenin expression, and serum exosomal miR-1246. In conclusion, our findings provided preclinical supports for ovatodiolide as a colon CSC inhibitor by reducing ß-catenin/STAT3/miR-1246 signaling conveyed by CSC derived exosomes.

Preclinical Evaluation of the Novel Small-Molecule MSI-N1014 for Treating Drug-Resistant Colon Cancer via the LGR5/ß-catenin/miR-142-3p Network and Reducing Cancer-Associated Fibroblast Transformation.

Colorectal cancer represents one of the most prevalent malignancies globally, with an estimated 140,000 new cases in the United States alone in 2019. Despite advancements in interventions, drug resistance occurs in virtually all patients diagnosed with late stages of colon cancer. Amplified epidermal growth factor receptor (EGFR) signaling is one of the most prevalent oncogenic drivers in patients and induces increased Janus kinase (JAK)/signal transduction and activator of transcription (STAT) and ß-catenin functions, all of which facilitate disease progression. Equally important, cancer-associated fibroblasts (CAFs) transformed by cancer cells within the tumor microenvironment (TME) further facilitate malignancy by secreting interleukin (IL)-6 and augmenting STAT3 signaling in colon cancer cells and promoting the generation of cancer stem-like cells (CSCs). Based on these premises, single-targeted therapeutics have proven ineffective for treating malignant colon cancer, and alternative multiple-targeting agents should be explored. Herein, we synthesized a tetracyclic heterocyclic azathioxanthone, MSI-N1014, and demonstrated its therapeutic potential both in vitro and in vivo. First, we used a co-culture system to demonstrate that colon cancer cells co-cultured with CAFs resulted in heightened 5-fluorouracil (5-FU) resistance and tumor sphere-forming ability and increased side populations, accompanied by elevated expression of cluster of differentiation 44 (CD44), ß-catenin, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and ATP-binding cassette super-family G member 2 (ABCG2). MSI-N1014 suppressed cell viability, colony formation, and migration in both DLD1 and HCT116 cells. MSI-N1014 treatment led to decreased expressions of oncogenic markers, including mammalian target of rapamycin (mTOR), EGFR, and IL-6 and stemness markers such as CD44, ß-catenin, and LGR5. More importantly, MSI-N1014 treatment suppressed the transformation of CAFs, and was associated with decreased secretion of IL-6 and vascular endothelial growth factor (VEGF) by CAFs. Furthermore, MSI-N1014 treatment resulted in significantly reduced oncogenic properties, namely the migratory ability, tumor-sphere generation, and resistance against 5-FU. Notably, an increased level of the tumor suppressor, miR-142-3p, whose targets include LGR5, IL-6, and ABCG2, was detected in association with MSI-N1014 treatment. Finally, we demonstrated the therapeutic potential of MSI-N1014 in vivo, where combined treatment with MSI-N1014 and 5-FU led to the lowest tumor growth, followed by MSI-N1014 only, 5-FU, and the vehicle control. Tumor samples from the MSI-N1014 group showed markedly reduced expressions of LGR5, ß-catenin, IL-6, and mTOR, but increased expression of the tumor suppressor, miR-142-3p, according to qRT-PCR analysis. Collectively, we present preclinical support for the application of MSI-N1014 in treating 5-FU-resistant colon cancer cells. Further investigation is warranted to translate these findings into clinical settings.

Erratum: Preclinical investigation of ovatodiolide as a potential inhibitor of colon cancer stem cells via downregulating sphere-derived exosomal ß-catenin/STAT3/miR-1246 cargoes.

[This corrects the article on p. 2337 in vol. 10, PMID: 32905416.].