Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis.

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Genetic disruption of Ano5 leads to impaired osteoclastogenesis for gnathodiaphyseal dysplasia.

OBJECTIVES: Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare skeletal genetic disorder characterized by sclerosis of tubular bones and cemento-osseous lesions in mandibles. TMEM16E/ANO5 gene mutations have been identified in patients with GDD. Here, Ano5 knockout (Ano5-/- ) mice with enhanced osteoblastogenesis were used to investigate whether Ano5 disruption affects osteoclastogenesis. SUBJECTS AND METHODS: The maturation of osteoclasts, formation of F-actin ring and bone resorption were detected by immunohistochemistry, TRAP, phalloidin staining and Coming Osteo assays. The expression of osteoclast-related factors was measured by qRT-PCR. Early signaling pathways were verified by western blot. RESULTS: Ano5-/- mice exhibited inhibitory formation of multinucleated osteoclasts with a reduction of TRAP activity. The expression of Nfatc1, c-Fos, Trap, Ctsk, Mmp9, Rank and Dc-stamp was significantly decreased in bone tissues and bone marrow-derived macrophages (BMMs) of Ano5-/- mice. Ano5-/- osteoclasts manifested disrupted actin ring and less mineral resorption. RANKL-induced early signaling pathways were suppressed in Ano5-/- osteoclasts and Ano5 knockdown RAW264.7 cells. Moreover, the inhibitory effects of NF-κB signalling pathway on osteoclastogenesis were partially attenuated with NF-κB signalling activator. CONCLUSIONS: Ano5 deficiency impairs osteoclastogenesis, which leads to enhanced osteogenic phenotypes mediated by bone homeostasis dysregulation.

Introduction of a Cys360Tyr Mutation in ANO5 Creates a Mouse Model for Gnathodiaphyseal Dysplasia.

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease characterized by the osteosclerosis of tubular bones and the formation of cemento-osseous lesions in mandibles. Although genetic mutations for GDD have been identified in the ANO5/TMEM16E gene, the cellular and molecular mechanisms behind the pathogenesis of GDD remain unclear. Here, we generated the first knock-in mouse model for GDD with the expression of human mutation p.Cys360Tyr in ANO5. Homozygous Ano5 knock-in mice (Ano5KI/KI ) replicated GDD-like skeletal features, including massive jawbones, bowing tibia, bone fragility, sclerosis, and cortical thickening of the femoral and tibial diaphysis. Serum alkaline phosphatase (ALP) levels were elevated in Ano5KI/KI mice as in GDD patients with p.Cys360Tyr mutation. Calvaria-derived Ano5KI/KI osteoblast cultures showed increased osteoblastogenesis, including hypermineralized bone matrix and enhanced bone formation-related factors expression. Interestingly, Ano5KI/KI bone marrow-derived macrophage cultures showed decreased osteoclastogenesis, and Ano5KI/KI osteoclasts exhibited disrupted actin ring formation, which may be associated with some signaling pathways. In conclusion, this new mouse model may facilitate elucidation of the pathogenesis of GDD and shed more light on its treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

Azoxystrobin Reduces Oral Carcinogenesis by Suppressing Mitochondrial Complex III Activity and Inducing Apoptosis.

PURPOSE: The five-year survival rate of patients with oral cancer is approximately 50%; thus, alternative drugs with higher efficacy are urgently required. Azoxystrobin (AZOX), a natural, novel methoxyacrylate fungicide isolated from mushrooms, has a broad-spectrum, with highly efficient bactericidal effect. However, studies on AZOX have focused on antifungal effects. Here, we explore the potential cancer-preventive effects of AZOX and the underlying mechanisms. MATERIALS AND METHODS: The effects of AZOX on oral carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO) were investigated in C57BL/6 mice. Cell proliferation and apoptosis were examined by Ki67 immunohistochemistry and TUNEL staining, respectively. The main organ coefficients of each group were calculated to evaluate the biosafety of AZOX. CCK8 and flow cytometry were used to detect the effects of AZOX on cell viability and apoptosis in oral cancer cell line CAL27 and SCC15 cells in vitro. Cell cycle, mitochondrial complex III activity, intercellular reactive oxygen species (ROS) level, mitochondrial ROS level, and mitochondrial membrane potential (MMP) were detected by flow cytometry in AZOX-treated CAL27 cells. RESULTS: AZOX significantly inhibited the occurrence of 4NQO-induced tongue cancer and delayed the progression of tongue precancerous lesions in mice. High-dose AZOX obviously inhibited cell viability and induced apoptosis in epithelial dysplastic and oral squamous cell carcinoma (OSCC) lesions in mouse tongue mucosa. AZOX was confirmed to have high biosafety. Similarly, in vitro cell viability was suppressed, and apoptosis was induced in AZOX-treated CAL27 and SCC15 cells. AZOX induced cell cycle arrest at the S phase. AZOX inhibited mitochondrial complex III activity, increased intracellular and mitochondrial ROS levels, and decreased MMP in CAL27 cells. CONCLUSION: AZOX inhibited the development of oral cancer through specific inhibition of the activity of mitochondrial complex III, which led to ROS accumulation, and MMP decrease, ultimately inducing apoptosis. AZOX may be a novel agent for the prevention and treatment of OSCC.

Chemopreventive effect of modified zengshengping on oral cancer in a hamster model and assessment of its effect on liver.

Ethnopharmacological relevance Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors, seriously compromising patients' quality of life. Previous studies showed that Zengshengping (ZSP), a popular traditional Chinese medicine, has certain inhibiting effects on both oral precancerous lesions and OSCC. However, few reports underlined ZSP side effects such as liver toxicity, which limit its long-term application. Aim of the study was to evaluate the chemopreventive effect of a modified ZSPs formula on oral cancer in a hamster model. Its effect on hamster liver was also assessed. Materials and Methods The original medicine (ZSP-1) and other two formulas slightly different and called ZSP-2 and ZSP-3 were prepared ahead of time. DMBA (0.5%) was topically applied for 6 weeks to induce a premalignant lesion on hamsters' cheek pouch, then ZSP-1/2/3 were intragastrically administered for 8 weeks. Hamster treated with DMBA + each of the ZSPs represented the ZSP-1/2/3 groups, while those without ZSP-1/2/3 treatment represented the DMBA group. To assess the effect of ZSPs in the liver, intragastric administration of ZSP-1/2/3 was carried out to other groups of hamsters for 12 weeks and the blood was collected every two weeks to detect the hepatic function. Some of the hamsters were sacrificed at the end of 12 weeks, while the remaining animals were sacrificed after other 4 weeks to estimate the effect of ZSP-1/2/3 withdrawal on the liver. Results showed that tumor development in the ZSP-1/2/3 groups was less than that in DMBA group. BrdU, CD31 and COX-2 expression in the hyperplastic tissues was significantly lower in the ZSP-1/2/3 groups than that in the DMBA group. In addition, VEGF and COX-2 expression in ZSP-1/2/3 groups was lower while caspase-9 and p53 expression was higher than those in the DMBA group. Finally, PTEN expression in ZSP-1/2/3 groups was higher than that in the DMBA group. As regard the effect in the liver, ALP in the ZSP-1/2/3 groups was higher than that in the control group treated with an intragastric administration of ddH2O. After 4 weeks of withdrawal, the hamsters of the ZSP-3 group did not recover from the increase in ALP. Histopathology showed the presence of inflammatory lesions in each group after 12 weeks, especially in the ZSP-1/3 groups, and the number of apoptotic cells in the ZSP-3 group was higher than that in the other groups, without any recovery after withdrawal of the drug. At 12 weeks, the MDA in the ZSP-1 group was higher than that in the control group and the ZSP-2 group, but the difference disappeared after drug withdrawal because the MDA in the ZSP-1/3 groups decreased. Conclusions ZSP-2 possessed a chemopreventive effect against oral cancer by inhibiting inflammation, proliferation of tumor cells, generation of microvessels and by promoting tumor cell apoptosis. In addition, hepatotoxicity of ZSP-2, which might be related to oxidative stress injury, was reduced to some extent.

The antilymphatic metastatic effect of hyaluronic acid in a mouse model of oral squamous cell carcinoma.

Objectives: Lymphatic metastasis is the main cause of low patient survival in cases of oral squamous cell carcinoma (OSCC). Several animal models have been established to uncover the mechanism that regulates lymph node metastasis of OSCC cells. Unfortunately, these models often take a long time to establish. The prolonged tumor burden can lead to animal cachexia, which may ultimately affect the experimental outcome. To overcome the disadvantages of these models, we established an orthotopic metastatic animal model of OSCC that showed quick lymph node metastasis potential.Results: DiR dye-labeled CAL27 cells were injected into tongue tissues of BALB/c nude mice, and the cells metastasized to lymph nodes on day 3. Metastasis was monitored using an in vivo imaging system and confirmed by histological observation. Using this model, we investigated the role of hyaluronic acid (HA) on the cervical metastasis of OSCC cells. Surprisingly, we found that the presence of HA significantly reduced the incidence of metastasis to cervical lymph nodes compared with the control group. Further analysis revealed that the presence of exogenous HA promoted mesenchymal-epithelial transition (MET) in primary tumors while reducing the metastatic potential of OSCC.Conclusion: Our findings confirmed the establishment of a fast and reliable lymphatic metastatic mouse model of OSCC that can be used for investigating metastatic mechanisms and analyzing various antimetastasis strategies. An equally important discovery is the antimetastatic property of HA, which could provide a potential therapeutic strategy for OSCC.

The Effect of Chemically Modified Tetracycline-3 on the Progression of Dental Caries in Rats.

OBJECTIVE: Matrix metalloproteinases (MMPs) exist in human saliva and dentin and play an important role in the degradation of organic matrix in teeth. Chemically modified tetracycline-3 (CMT-3) is an inhibitor of MMPs. CMT-3 has been used experimentally to treat caries since 1999, but no distinction between dental caries prevalence and dentin caries prevalence has been described. METHODS: A total of 65 Sprague-Dawley rats were randomly divided into three groups. The positive control group (25 rats) was inoculated with Streptococcus mutans (ATCC700610) and fed the cariogenic feed of improved Keyes Diet 2000. The CMT-3 group (25 rats) was also inoculated with S. mutans and fed the cariogenic feed of improved Keyes Diet 2000; the surfaces of rats' molars were daily treated with 0.02% CMT-3. The negative control group (15 rats) was only fed the standard rodent chow. At the end of the 10th week, the dental caries prevalence and dentin caries prevalence of each group were calculated, and the regions of caries were assessed. RESULTS: No caries was found in the negative control group. The dental caries prevalence of the CMT-3 and the positive control group was 75.0 and 83.3%, respectively (p > 0.05, Table 2). The dentin caries prevalence of the CMT-3 and the positive control group was 33.3 and 70.8%, respectively (p < 0.05, Table 2). The Keyes scoring of dentin caries in the CMT-3 group was significantly lower than that in the positive control group (p < 0.05, Table 3). CONCLUSIONS: CMT-3 had no effect on the prevalence of dental caries, but could lower the prevalence and slow down the progression of dentin caries.

Anti-tumor Effect of Rhaponticum uniflorum Ethyl Acetate Extract by Regulation of Peroxiredoxin1 and Epithelial-to-Mesenchymal Transition in Oral Cancer.

Objective: To explore whether Rhaponticum uniflorum (R. uniflorum) had anti-tumor effects in oral cancer and investigate the molecular mechanisms involved in these anti-tumor effects. Methods: Chemical compositions of R. uniflorum ethyl acetate (RUEA) extracts were detected by ultra-performance liquid chromatography-Q/time-of-flight mass spectrometry (UPLC-Q/TOF-MS), followed by pharmacology-based network prediction analysis. The effects of RUEA extracts on proliferation, apoptosis, migration, and invasion ability of human oral squamous cell carcinoma (OSCC) cell line SCC15 were evaluated by CCK8 assay, Annexin V- fluorescein isothiocyanate/propidium iodide staining, wound healing assay, and Matrigel invasion assay, respectively. The mRNA and protein expression of peroxiredoxin1 (Prx1), the epithelial-to-mesenchymal transition (EMT) marker E-cadherin, vimentin, and Snail were determined by quantitative real-time reverse transcription polymerase chain reaction and western blotting. A mouse xenograft model of SCC15 cells was established to further evaluate the effect of RUEA extracts in vivo. Immunohistochemical assessment of Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic cells were performed on the tumor tissues to assess the effects of RUEA extracts on proliferation and apoptosis. Results: Fourteen compounds were identified from RUEA extracts by UPLC-Q/TOF-MS. The pharmacology-based network prediction analysis showed that Prx1 could be a potential binder of RUEA extracts. In SCC15 cells, RUEA extracts inhibited cell viability, induced apoptosis, and suppressed cell invasion and migration in a concentration-dependent manner. After treatment with RUEA extracts, the mRNA and protein expression of E-cadherin increased, whereas those of Prx1, vimentin, and Snail decreased. RUEA extracts also affected the EMT program and suppressed cell invasion and migration in Prx1 knockdown SCC15 cells. In an OSCC mouse xenograft model, RUEA extracts (25 and 250 mg/kg) significantly inhibited the growth of tumors. Compared with the control group, Ki67 expression was reduced and apoptosis rates were elevated in the transplanted tumors treated with RUEA extracts. RUEA extracts increased the expression of E-cadherin and decreased the expression of Prx1, vimentin, and Snail in vivo. Conclusion: RUEA extracts inhibited tumor growth and invasion by reducing Prx1 expression and suppressing the EMT process in OSCC. RUEA extracts may be a potential candidate for OSCC treatment.

Nicotine suppresses apoptosis by regulating α7nAChR/Prx1 axis in oral precancerous lesions.

Nicotine, a tumor promoter in tobacco, can increase Peroxiredoxin (Prx1) and nicotinic acetylcholine receptors (nAChRs) in oral squamous cell carcinoma (OSCC). In the present study, we investigate the effects of nicotine in oral precancerous lesions focusing on apoptosis and nAChR/Prx1 signaling. We detected expression of Prx1, α3nAChR, α7nAChR, phosphorylation of mitogen-activated protein kinases (MAPK) and apoptosis in dysplastic oral keratinocyte (DOK) cells as well as in 4-nitroquinoline 1-oxide (4NQO) or 4NQO + nicotine - induced oral precancerous lesions in Prx1 wild-type (Prx1+/+) and Prx1 knockdown (Prx1+/-) mice. In DOK cells, Prx1 knockdown and blocking α7nAChR activated apoptosis, and nicotine increased the expression of Prx1, α3nAChR and α7nAChR, and inhibited MAPK activation. Moreover, nicotine suppressed apoptosis depending on Prx1 and α7nAChR in DOK cells. In animal bioassay, nicotine and Prx1 promoted growth of 4NQO-induced precancerous lesions in mouse tongue. 4NQO plus nicotine suppressed MAPK activation in Prx1 wild-type mice but not in Prx1 knockdown mice. Our data demonstrate that nicotine inhibits cell apoptosis and promotes the growth of oral precancerous lesions via regulating α7nAChR/Prx1 during carcinogenesis of OSCC.

Involvement of the Nonneuronal Cholinergic System in Bone Remodeling in Rat Midpalatal Suture after Rapid Maxillary Expansion.

Few studies sought to analyze the expression and function of the nonneuronal acetylcholine system in bone remodeling in vivo due to the lack of suitable models. We established a rat maxilla expansion model in which the midline palatine suture of the rat was rapidly expanded under mechanical force application, inducing tissue remodeling and new bone formation, which could be a suitable model to investigate the role of the nonneuronal acetylcholine system in bone remodeling in vivo. During the expansion, the expression pattern changes of the nonneuronal cholinergic system components and the mRNA levels of OPG/RANKL were detected by immunohistochemistry or real-time PCR. The value of the RANKL/OPG ratio significantly increased after 1 day of expansion, indicating dominant bone resorption induced by the mechanical stimulation; however after 3 days of expansion, the value of the RANKL/OPG ratio significantly decreased, suggesting a dominant role of the subsequent bone formation process. Increasing expression of Ach was detected after 3 days of expansion which indicated that ACh might play a role in bone formation. The mRNA expression levels of other components also showed observable changes during the expansion which confirmed the involvement of the nonneuronal cholinergic system in the process of bone remodeling in vivo. Further researches are still needed to figure out the detailed functions of the nonneuronal cholinergic system and its components.

[Chemopreventive effect of boswellic acid and curcumin on 7,12-dimethyl benzanthracene-induced hamster cheek pouch carcinogenesis].

OBJECTIVE: To evaluate the chemopreventive effects of boswellic acid and curcumin on 7,12-dimethyl benzanthracene(DMBA)-induced oral carcinogenesis in the hamster cheek pouch model. METHODS: Male Syrian golden hamsters (6 - 8 weeks old, 80 - 130 g in weight) were randomly divided into seven groups, with group A serving as the untreated negative control. The left cheek pouch of the remaining hamsters was topically treated with 0.5% DMBA in mineral oil three times a week for 6 weeks. They were then randomized to six groups with group B serving as a positive control and receiving no further treatment. Groups C-G were treated topically with 5, 10 mg/L boswellic acid, 5, 10 µmol/L curcumin, or the combination of 5 mg/L boswellic acid and 5 µmol/L curcumin three times per week for 18 weeks. The animals were injected with bromodeoxyuridine intraperitoneally at 50 mg/kg 2 h prior to killing. At the 25 th week all the hamsters were sacrificed and cheek pouch tissue was harvested. One half of the tissue was snap frozen in liquid nitrogen for analysis of arachidonic acid metabolites, and the other half was fixed in 10% phosphate-buffered saline(PBS)-buffered formalin for histopathological examination. RESULTS: Six-weeks of DMBA followed by 18-weeks of topical application of boswellic acid and curcumin, both boswellic acid (5, 10 mg/L) and curcumin (5, 10 µmol/L) significantly inhibited the incidence from 93.8% to 73.9% (P > 0.05), numbers from 2.19 ± 0.98 to 1.13 ± 0.81 (P < 0.01) and size of visible tumors. Microscopically the incidence of squamous cell carcinoma and BrdU index were also significantly suppressed by boswellic acid and curcumin. CONCLUSIONS: Both boswellic acid and curcumin were effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model.