Search Results (43)

Background/Objectives: This study aimed to explore the potential value of narrow-band-imaging (NBI)-derived mean optical intensity (MOI) in monitoring the progression of oral squamous cell carcinoma (OSCC), from the normal oral mucosa through epithelial dysplasia to invasive carcinoma. We compared differences in the NBI MOI among distinct pathological stages, so as to provide preliminary evidence for its clinical application in auxiliary diagnosis and progression assessment for OSCC. Methods: A total of 40 human oral mucosal specimens (15 normal, 15 oral leukoplakia, 10 OSCC) were enrolled for NBI image acquisition and MOI measurements. A 4-nitroquinoline-1-oxide (4NQO)-induced mouse OSCC model (n = 34) was used to dynamically record MOI changes across different pathological stages. A syngeneic tongue tumor mouse model (n = 16) was further established to evaluate whether MOI could reflect tumor formation and growth. All MOI values were quantified using ImageJ software with standardized region-of-interest (ROI) selection and background correction. Results: In clinical samples, MOI values decreased progressively from the normal mucosa (129.6 ± 5.991 arbitrary units (a.u.)) to oral leukoplakia (OLK) subgroups, including mild dysplasia (104.6 ± 3.757 a.u.) and moderate-to-severe dysplasia (91.77 ± 4.345 a.u.), and further to OSCC (54.41 ± 14.40 a.u.). In the 4NQO model, the MOI of the lingual mucosa was highest in the healthy control group (167.3 ± 10.05 a.u.) and gradually declined with increasing dysplasia severity, reaching the lowest level at the OSCC stage (48.67 ± 10.07 a.u.). In the syngeneic tumor model, the MOI was significantly lower in tumor-bearing mice than in healthy controls (47.85 ± 10.44 a.u. vs. 119.7 ± 14.20 a.u., p < 0.001). Receiver operating characteristic (ROC) analysis demonstrated good diagnostic performance of the MOI in distinguishing healthy tissue from cancerous lesions. Conclusions: NBI-derived MOI may quantitatively reflect the dynamic alterations of the oral mucosa during oral carcinogenesis and could represent a potential biomarker enabling the non-invasive, repeatable early evaluation and dynamic monitoring of OSCC. Full article

Rationale: Oral squamous cell carcinoma (OSCC) carries a poor prognosis despite advances in multimodal therapy. Nanomedicine represents a compelling strategy to enhance targeted drug delivery and improve therapeutic outcomes. Here, we investigated sEV-mediated delivery of curcumin as a novel therapeutic approach for OSCC. Methods: Small extracellular vesicles (sEVs) were isolated from Jurkat cells by size-exclusion chromatography and loaded with curcumin via sonication to generate JCsEV. Functional effects were assessed in vitro using wound healing, transwell invasion, and metabolic activity assays across multiple cancer cell lines. Therapeutic efficacy in vivo was evaluated in the 4-nitroquinoline 1-oxide (4-NQO) immunocompetent murine model of oral carcinogenesis. Female C57BL/6J mice received intraperitoneal treatment for four weeks with PBS, free curcumin, unloaded JsEV, or JCsEV. Tumor number, tumor burden, and body weight changes were assessed at the experimental endpoint. Results: In vitro, JCsEV significantly inhibited tumor cell migration, invasion, and metabolic activity compared with controls (p < 0.05). In vivo, treatment with JCsEV significantly reduced tumor number and tumor burden in the 4-NQO model (p < 0.01). In addition, body weight loss was reduced in JCsEV-treated mice compared with controls. Conclusion: sEV-mediated delivery of curcumin effectively suppresses tumor progression in experimental OSCC. These findings establish proof-of-concept for sEV-based nanomedicine as a therapeutic strategy for OSCC and provide a compelling rationale for further translational investigation of sEVs as drug delivery platforms. Full article

Background: Squamous cell carcinoma of head and neck (SCCHN) is a devastating disease with high morbidity and mortality and the 6th most common cancer worldwide. The 5-year relative survival for advanced-stage disease is below 50%, stressing the need for chemoprevention. In the current study, we investigated the chemopreventive efficacy of the combination of resveratrol and epigallocatechin gallate (EGCG). Methods: We used the 4-Nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis model. C57BL/6 mice were exposed to drinking water containing 4-NQO for 10 weeks. From week 11, mice were treated with vehicle, resveratrol, EGCG and their combination until week 22. RNASeq, qPCR and in silico analysis were performed identifying differentially expressed genes and enriched pathways. Results: Resveratrol alone and in combination with EGCG significantly inhibited the number of visible lesions, whereas the number of microscopic lesions and lesion areas were significantly inhibited only by the combination. The expression of Ki-67 was also significantly inhibited in resveratrol and combination groups. Growth differentiation factor 15 (GDF15), Activation transcription factor 3 (ATF3) and several other genes associated with xenobiotic metabolism as significantly upregulated genes, with GDF15 being the most upregulated one. Furthermore, hallmarks of xenobiotic metabolism and several other anticancer pathways were enriched after treatment with resveratrol and the combination. Conclusions: Our data strongly demonstrate the chemopreventive potential of the combination of resveratrol and EGCG and pave the way for further clinical developments. Full article

Purpose: Due to the lack of effective local therapeutic strategies for oral squamous cell carcinoma (OSCC), this study aimed to develop a novel gelatin/lignin hydrogel loaded with mesenchymal stem cell (MSC)-derived exosomes enriched in microRNA-185 (miR-185 EV) for intraoral delivery, followed by systematic evaluation of its therapeutic efficacy and underlying molecular mechanisms. Materials and Methods: The gelatin/lignin hydrogel was prepared and subsequently loaded with miR-185 EV. The physicochemical properties of the hydrogel, including microstructure, swelling behavior, chemical composition, and rheological characteristics, were systematically evaluated. Next, the stability, viscosity, biocompatibility, and exosome release kinetics of the hydrogel were further assessed. A 4-nitroquinoline-1-oxide (4NQO)-induced mouse tongue carcinogenesis model was established to assess the in vivo antitumor activity of the hydrogel via intraoral administration. Moreover, a proteomic analysis was conducted to investigate the molecular mechanisms of miR-185 EV on OSCC. Results: The miR-185 EV-loaded gelatin/lignin hydrogel exhibited favorable physicochemical properties, stability, and biocompatibility while prolonging the tissue retention time of miR-185 EV. In vivo antitumor efficacy experiments showed that the miR-185 EV-loaded hydrogel significantly inhibited tumor occurrence and alleviated epithelial dysplasia. Immunohistochemical analyses revealed significant suppression of tumor proliferation and epithelial–mesenchymal transition (EMT) of the hydrogel. Proteomic analysis indicated that miR-185 EV suppressed OSCC progression by downregulating interleukin-1β (IL-1β), consequently inhibiting the NF-κB signaling pathway. Conclusion: The findings demonstrate the successful development of the miR-185 EV-loaded gelatin/lignin hydrogel that represents an effective nanomedicine platform for intraoral drug delivery, providing a promising strategy for the clinical treatment of OSCC. Full article

Objectives: Tobacco has been associated with the development of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). This study aimed to evaluate the in vitro and in vivo changes caused by carcinogen 4-nitroquinoline 1-oxide (4NQO), simulating smoking conditions. Materials and Methods: In the in vitro study, normal keratinocytes were exposed to 1.3 µM and 2.6 µM concentrations of 4NQO to induce dysplastic transformation (H-DISP) and malignant transformation (H-SCC), respectively. The cells were collected and subjected to hematoxylin and eosin (H&E) staining and immunocytochemistry with Ki-67. For the in vivo study, female C57BL/6J mice were divided into a pure control (PC) group and experimental groups exposed to 50 µg/mL (NQ) and 100 µg/mL (CM) of 4NQO in autoclaved drinking water. Each group was euthanized after 8, 12, 16, and 20 weeks of exposure. The tongues were collected, processed, stained with H&E, and analyzed using conventional light microscopy. Results: In vitro, significant morphological changes were observed in the H-DISP and H-SCC groups, with a cell proliferation index exceeding 30% in the H-DISP group. In vivo, the CM group showed greater progression to severe dysplasia/carcinoma within a shorter treatment period compared to the NQ group. Conclusions: We established critical doses and exposure durations for 4NQO, both in vitro and in vivo, to induce cellular changes and the formation of OL and OSCC, providing a standardized model for studies related to oral carcinogenesis. Full article

The mutagenic potential of coffee by-products, including Coffea leaves, blossoms, cherries, and silverskin, was studied using thin-layer chromatography (TLC) coupled with the recent planar Ames bioassay via pH indicator endpoint. The 2LabsToGo-Eco allowed for the separation and detection of mutagens in complex samples. Hot water was the most effective extraction solvent in terms of yield and closely simulated the typical human consumption of coffee by-products. Separation was performed on TLC plates with a mixture of ethyl acetate, n-propanol, and water, followed by bioassay detection. The positive control 4-nitroquinoline 1-oxide exhibited clear mutagenic responses, confirming the proper bioassay performance. In the Ames bioautogram, none of the tested coffee by-products showed mutagenic zones, suggesting the absence of strongly acting, acute mutagens under the applied test conditions; however, given the only 5 h short incubation and the use of TA98 strain only, a longer incubation time and testing with additional Salmonella strains is recommended. The results provide new safety data for Coffea leaves and blossoms and are consistent with some previous studies demonstrating the safety of coffee by-products. However, further improvements in the sensitivity and selectivity of the planar Ames bioassay are demanded, and further in vivo and long-term safety studies are recommended. Considering natural variability, the different uses of pesticides and treatments, and the fluctuating supply chains, coffee by-products may differ highly. The planar bioassay technology using the affordable 2LabsToGo-Eco is a powerful toxicological screening option for the coffee industry, considering the increasing interest in utilizing coffee by-products. Full article

Oral leukoplakia (OL) is the most common potentially malignant oral disorder, with variable risk of progression to oral squamous cell carcinoma (OSCC). This study evaluated the chemopreventive and immunomodulatory potential of Artemisinin (ART) and Rubus occidentalis (RO), alone or combined (ARO), in a 4NQO-induced murine model. Mice received 4NQO (100 µg/mL) in drinking water, and treatments began at week 8. Animals were euthanized at weeks 12 and 16 for histological, apoptotic (caspases-3, -8, -9; calreticulin), inflammatory (IL-1β, IL-10, HMGB1), and immune (CD8, CD68, CD56, IFN-γ, GM-CSF) marker analyses. RO-treated animals showed delayed malignant transformation, with no carcinomas at week 16 and increased expression of caspase-9, calreticulin, HMGB1, IFN-γ, and GM-CSF, indicating transient activation of antitumor immune responses. ART-treated mice showed increased CD68 and reduced CD56 expression, suggesting an immunosuppressive profile and higher carcinoma incidence. The ARO combination did not improve outcomes beyond ART alone. These findings support the immunomodulatory and pro-apoptotic effects of RO in delaying OL progression, highlighting its chemopreventive potential. ART showed limited benefit under current conditions, warranting further investigation into dose optimization and synergistic strategies. Full article

Background/Objectives: Oral squamous cell carcinoma (OSCC) is the most common oral cancer, progressing from hyperplasia to dysplasia, carcinoma in situ (CIS), and finally invasive squamous cell carcinoma (ISCC). Developing an animal model that mimics both early and advanced OSCC stages has been challenging. The 4-Nitroquinoline 1-oxide (4NQO) model is considered one of the most suitable, as it represents all stages of OSCC. Nevertheless, thoroughly understanding the properties of the 4NQO model is essential for preclinical testing of novel therapeutics. Methods: We aimed to characterize the 4NQO rat model using two application methods—drinking water and topical application—over eight months. Monthly sacrifices allowed histopathological analysis and ex vivo magnetic resonance imaging (MRI) to track tumor progression. Results: CIS was observed at three months in the drinking water group, evolving into ISCC by six months, while topical application induced CIS at eight months without ISCC formation. The tongue was divided into three regions and histological properties, lesion size, and invasion depth were analyzed. In the drinking water group, particularly in the body of the tongue, we saw earlier CIS development, larger lesions, and deeper invasion. Additionally, assessment of proliferative properties showed an increased cell division in dysplastic lesions that reduced upon invasion. MRI was able to show macroscopic tumoral lesions, in concordance with histology. Conclusions: Overall, the drinking water method closely mimics human OSCC, validating the 4NQO model for translational OSCC research. Full article

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25–30% survival of larvae compared with parental strain. Full article

Background and Objectives: PIN1 is overexpressed in several human cancers, including prostate cancer, breast cancer, and oral squamous carcinomas. Juglone (J), derived from walnut, was reported to selectively inhibit PIN1 by modifying its sulfhydryl groups. In this study, the potential effects of juglone, also known as PIN1 inhibitor, on oral cancer and carcinogenesis were investigated at the molecular level. Materials and Methods: 4-Nitroquinoline N-oxide (4-NQO) was used to create an oral cancer model in animals. Wistar rats were divided into five groups: Control, NQO, Juglone, NQO+J, and NQO+J*. The control group received the basal diet and tap water throughout the experiment. The NQO group received 4-NQO for 8 weeks in drinking water only. The Juglone group was administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). The NQO+J group received 4-NQO in drinking water for 8 weeks, starting 1 week after the cessation of 4-NQO treatment. They were then administered intraperitoneally in a juglone solution for 10 weeks. (1 mg/kg/day). NQO+J* group: received 4 NQO for 8 weeks in drinking water and administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). They were sacrificed at the end of the 22-week experimental period. The tongue tissues of the rats were isolated after the experiment, morphological changes were investigated by histological examinations, and the molecular apoptotic process was investigated by rt-qPCR and western blot. Results: Histological results indicate that tumors are formed in the tongue tissue with 4-NQO, and juglone treatment largely corrects the epithelial changes that developed with 4-NQO. It has been determined that apoptotic factors p53, Bax, and caspases are induced by the effect of juglone, while antiapoptotic factors such as Bcl-2 are suppressed. However, it was observed that the positive effects were more pronounced in rats given juglone together with 4-NQO. Conclusions: The use of PIN1 inhibitors such as juglone in place of existing therapeutic approaches might be a promising and novel approach to the preservation and treatment of oral cancer and carcinogenesis. However, further research is required to investigate the practical application of such inhibitors. Full article

4-Nitroquinoline-N-oxide (NQO) and 4-nitropyridine-N-oxide (NPO) are important precursors for the synthesis of substituted heterocycles while NQO is a popular model mutagen and carcinogen broadly used in cancer research; intermolecular interactions are critical for their reactions or functioning in vivo. Herein, the effects of the coordination of N-oxide’s oxygen atom to Lewis acids on multicenter donor–acceptor bonding were explored via a combination of experimental and computational studies of the complexes of NQO and NPO with a typical π-electron donor, pyrene. Coordination with ZnCl₂ increased the positive electrostatic potentials on the surfaces of these π-acceptors and lowered the energy of their LUMO. Analogous effects were observed upon the protonation of the N-oxides’ oxygen or bonding with boron trifluoride. The interaction of ZnCl₂, NPO, or NQO and pyrene resulted in the formation of dark co-crystals comprising π-stacked Zn-coordinated N-oxides and pyrene similar to that found with protonated or (reported earlier) BF₃-bonded N-oxides. Computational studies indicated that the coordination of N-oxides to zinc(II), BF₃, or protonation led to the strengthening of the multicenter bonding of the nitro-heterocycle with pyrene, and this effect was related both to the increased electrostatic attraction and molecular–orbital interactions in their complexes. Full article

Copper and zinc organometallics have multiple applications and many are considered “data-poor” because the available toxicological information is insufficient for comprehensive health risk assessments. To gain insight into the chemical prioritization and potential structure activity relationship, the current work compares the in vitro toxicity of nine “data-poor” chemicals to five structurally related chemicals and to positive DNA damage inducers (4-nitroquinoline-oxide, aflatoxin-B1). The HC-04 non-cancer human liver cell line was used to investigate the concentration–response effects (24 h and 72 h exposure) on cell proliferation, DNA damage (γH2AX and DNA unwinding assays), and epigenetic effects (global genome changes in DNA methylation and histone modifications using flow cytometry). The 24 h exposure screening data (DNA abundance and damage) suggest a toxicity hierarchy, starting with copper dimethyldithiocarbamate (CDMDC, CAS#137-29-1) > zinc diethyldithiocarbamate (ZDEDC, CAS#14324-55-1) > benzenediazonium, 4-chloro-2-nitro-, and tetrachlorozincate(2-) (2:1) (BDCN4CZ, CAS#14263-89-9); the other chemicals were less toxic and had alternate ranking positions depending on assays. The potency of CDMDC for inducing DNA damage was close to that of the human hepatocarcinogen aflatoxin-B1. Further investigation using sodium-DMDC (SDMDC, CAS#128-04-1), CDMDC and copper demonstrated the role of the interactions between copper and the DMDC organic moiety in generating a high level of CDMDC toxicity. In contrast, additive interactions were not observed with respect to the DNA methylation flow cytometry data in 72 h exposure experiments. They revealed chemical-specific effects, with hypo and hypermethylation induced by copper chloride (CuCl₂, CAS#10125-13-0) and zinc-DMDC (ZDMDC, CAS#137-30-4), respectively, but did not show any significant effect of CDMDC or SDMDC. Histone-3 hypoacetylation was a sensitive flow cytometry marker of 24 h exposure to CDMDC. This study can provide insights regarding the prioritization of chemicals for future study, with the aim being to mitigate chemical hazards. Full article

Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity. Full article

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals. Full article

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents. Full article