TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC), the predominant malignancy of the oral cavity, is characterized by high incidence and low survival rates. Emerging evidence suggests a link between circadian rhythm disruptions and cancer development. The circadian gene TIMELESS, known for its specific expression in various tumors, has not been extensively studied in the context of OSCC. This study aims to explore the influence of TIMELESS on OSCC, focusing on cell growth and metabolic alterations. METHODS: We analyzed TIMELESS expression in OSCC using western blot, immunohistochemistry, qRT-PCR, and data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The role of TIMELESS in OSCC was examined through clone formation, MTS, cell cycle, and EdU assays, alongside subcutaneous tumor growth experiments in nude mice. We also assessed the metabolic impact of TIMELESS by measuring glucose uptake, lactate production, oxygen consumption, and medium pH, and investigated its effect on key metabolic proteins including silent information regulator 1 (SIRT1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), recombinant lactate dehydrogenase A (LDHA) and glucose transporter-1 (GLUT1). RESULTS: Elevated TIMELESS expression in OSCC tissues and cell lines was observed, correlating with reduced patient survival. TIMELESS overexpression enhanced OSCC cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). Conversely, TIMELESS knockdown inhibited these cellular and metabolic processes, an effect mirrored by manipulating SIRT1 levels. Additionally, SIRT1 was positively associated with TIMELESS expression. The expression of SIRT1, HK2, PKM2, LDHA and GLUT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS. CONCLUSION: TIMELESS exacerbates OSCC progression by modulating cellular proliferation and metabolic pathways, specifically by enhancing glycolysis and reducing oxidative phosphorylation, largely mediated through the SIRT1 pathway. This highlights TIMELESS as a potential target for OSCC therapeutic strategies.

Long non-coding RNA PTCSC3 inhibits human oral cancer cell proliferation by inducing apoptosis and autophagy.

INTRODUCTION: Long non-coding RNAs (lncRNAs) have gained increased attention due to the discovery of their roles in cancer-related processes. LncRNA PTCSC3 has been shown to have tumour-suppressive effects in thyroid cancer and glioblastoma. This study investigated the role of lncRNA PTSC3 in human oral cancer. MATERIAL AND METHODS: Cell viability was determined by MTT assay. The induction of apoptosis was confirmed by 4',6-diamidino-2-phenylindole (DAPI) and Annexin V/PI assays. Ultrastructural analysis was performed by electron microscopy. Transwell assay was used to monitor the invasion of oral cancer cells. RESULTS: The results revealed significant (p < 0.05) suppression of PTCSC3 expression in human oral cancer tissues and cell lines. The overexpression of PTCSC3 caused a significant (p < 0.05) decline in the proliferation of the human oral cancer cells via induction of apoptotic cell death which was accompanied by remarkable enhancement of Bax and suppression of Bcl-2. The electron microscopic analysis showed the development of autophagic vesicles in both the SCC-1 and SCC-9 cells indicative of autophagy. The western blotting analysis showed that PTCSC3 overexpression caused a remarkable increase in LC3B-I and Beclin 1 expression. PTCSC3 overexpression caused a significant (p < 0.05) decrease in invasion of the human SCC-1 and SCC-9 oral cancer cells. The invasion of the SCC-1 and SCC-9 cells was inhibited by 62% and 69% respectively. CONCLUSIONS: Overall, the evidence suggests that lncRNA PTCSC3 acts as a tumour suppressor in human oral cancer and suppresses oral cancer proliferation via induction of apoptosis and autophagy.

Response of mandibular condyles of juvenile and adult rats to abnormal occlusion and subsequent exemption.

OBJECTIVE: The adaptation capacities of the mandibular condyle in response to mechanical stimuli might be different between juveniles and adults, but has not been compared. This study aimed to investigate whether abnormal molar occlusion and subsequent molar extraction could lead to different remodeling responses in the mandibular condyles of juvenile and adult rats. METHODS: Abnormal molar occlusion (AMO) was established in the 5- and 16-wk old rats by moving their maxillary left and mandibular right third molars distally. AMO was removed in the molar extraction group at 4 weeks but remained in the AMO group. All rats were sacrificed at 8 weeks. Micro-computed tomography, histomorphology, immunohistochemistry and real-time PCR were adopted to evaluate the remodeling of condylar subchondral bone. RESULTS: Condylar subchondral bone loss and increased osteoclastic activities were observed in both juvenile and adult AMO groups, while increased osteoblastic activities were only seen in the juvenile AMO group. Decreased bone mineral density, bone volume fraction and trabecular thickness, but increased trabecular separation, number and surface of osteoclasts and mRNA levels of TRAP, cathepsin-K, RANKL in the juvenile AMO group were all reversed after molar extraction (all P<0.05). However, these parameters showed no difference between adult AMO and extraction groups (all P>0.05). CONCLUSIONS: Abnormal molar occlusion led to degenerative remodeling in the mandibular condyles of both juvenile and adult rats, while exemption of abnormal occlusion caused significant rescue of the degenerative changes only in the juvenile rats.

Betulinic Acid Inhibits Cell Proliferation in Human Oral Squamous Cell Carcinoma via Modulating ROS-Regulated p53 Signaling.

Oral squamous cell carcinoma (OSCC) is a common cancer of the head and neck. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid. The present study was designed to explore the effects of BA on OSCC KB cell proliferation in vitro and on implanted tumor growth in vivo and to examine the possible molecular mechanisms. The results showed that BA dose-dependently inhibited KB cell proliferation and decreased implanted tumor volume. In addition, BA significantly promoted mitochondrial apoptosis, as reflected by an increase in TUNEL+ cells and the activities of caspases 3 and 9, an increase in Bax expression, and a decrease in Bcl-2 expression and the mitochondrial oxygen consumption rate. BA significantly increased cell population in the G0/G1 phase and decreases the S phase cell number, indicating the occurrence of G0/G1 cell cycle arrest. ROS generation was significantly increased by BA, and antioxidant NAC treatment markedly inhibited the effect of BA on apoptosis, cell cycle arrest, and proliferation. BA dose-dependently increased p53 expression in KB cells and implanted tumors. p53 reporter gene activity and p53 binding in the promoters of Bax were significantly increased by BA. Knockdown of p53 blocked BA-induced increase in apoptosis, cell cycle arrest, and inhibition of cell proliferation. NAC treatment suppressed BA-induced increase in p53 expression. Furthermore, phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased by BA. Taken together, the data demonstrated that ROS-p53 signaling was crucial for BA-exhibited antitumor effect in OSCC. BA may serve as a potential drug for the treatment of oral cancer.

MicroRNA-27a-3p regulates epithelial to mesenchymal transition via targeting YAP1 in oral squamous cell carcinoma cells.

MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. Here, we profiled isolated cells from freshly resected tumors from oral squamous cell carcinoma (OSCC) patients and OSCC cell lines using a SYBR Green-based qPCR miRNA array to identify the expression change of the miRNAs. Based on the microarray data and clincopathological factor analysis of 50 OSCC patients related to these miRNAs, miR-27a-3p was selected as a putative miRNA which might play important role in OSCC progression. By bioinformatics analysis and dual-luciferase reporter assay, we found that YAP1 (Yes-associated protein-1) was a direct target gene of miR-27a-3p. Intriguingly, increased expression of miR-27a-3p could significantly decrease the expression level of YAP1 as well as several epithelial to mesenchymal transition (EMT)-related molecules in OSCC cell lines, including Twist and Snail. Then, follow-up studies revealed that miR-27a-3p expression was able to downregulate the EMT-related molecules effectively, which might be involved in the regulation of Sox2 via the YAP1-OCT4-Sox2 signaling axis. In summary, this study found that miR-27a-3p could inhibit the YAP1 directly by post-transcriptionally silencing and potentially suppress EMT process, suggesting that miR­27a-3p might play pivotal roles in effectively manipulating the invasion and metastasis in oral squamous cell carcinoma cells through the EMT inhibition.

A polysaccharide from the alkaline extract of Glycyrrhiza inflata induces apoptosis of human oral cancer SCC-25 cells via mitochondrial pathway.

In the present study, we isolated and characterized a homogenous polysaccharide (GIAP1) from the alkaline extract of the roots of Glycyrrhiza inflata. The anti-tumor activity of GIAP1 toward human oral cancer SCC-25 cells and the underlying mechanisms were also examined in vitro. GIAP1 dose-dependently inhibited the proliferation of SCC-25 cells via inducing apoptosis. Moreover, GIAP1 downregulated Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), and caused the release of cytochrome c to cytosol. Besides, GIAP1 triggered activation of capase-3 and caspase-9, as well as the degradation of poly (ADP-ribose) polymerase (PARP). In addition, the caspase-3 or caspase-9 inhibitor significantly inhibited GIAP1-induced apoptosis in SCC-25 cells. Collectively, we can conclude that the GIAP1 induces apoptosis in SCC-25 cells via a mitochondrial pathway.

Licochalcone A as a potent antitumor agent suppresses growth of human oral cancer SCC-25 cells in vitro via caspase-3 dependent pathways.

The majority of anticancer drugs are of natural origin. However, it is unknown whether licochalcone A is cytotoxic towards oral squamous cell carcinoma (OSCC) cells. The goal of this study was to investigate the cytotoxic effects of licochalcone A on the human OSCC SCC-25 cells and to identify the underlying molecular mechanism. Exposure of SCC-25 cells to licochalcone A dose- and time-dependently decreased cell viability by arresting cell cycle at the S and G2/M phase as well as inducing apoptosis. Furthermore, the proapoptotic activity of licochalcone A was revealed by DNA fragmentation. Concomitantly, we observed activation of the effector caspases-3, induced by activation of the initiator caspases -8 and -9, which subsequent trigger both death receptor pathway and the mitochondrial apoptotic pathway in licochalcone A-mediated SCC-25 cell apoptosis. Besides, treatment with 50 µg/mL of licochalcone A for 36 h led to the cleavage of PARP, an indicator of apoptosis induction. Therefore licochalcone A may be a good candidate for development as a possible chemopreventive agent against OSCC.

Treatment of infected soft tissue blast injury in swine by regulated negative pressure wound therapy.

OBJECTIVE: This study was designed to investigate the therapeutic potential of regulated negative pressure wound therapy (RNPT) in treating infected blast injuries in swine. BACKGROUND: Approximately 30% to 80% of blast injuries develop infection, which increases the morbidity and mortality of these casualties. RNPT has been used in US military operations in Iraq; however, no randomized controlled study has been conducted on the use of RNPT to treat infected war injuries. METHODS: Infected soft tissue blast injuries were treated with gauze dressings or RNPT with different pressures ranging from -5 to -35 kPa. To evaluate the wound healing process, the wound area, wound depth, the number of proliferative cells, and the vascular endothelial cells in the granulation tissue were measured at different time points. Furthermore, to evaluate the infection and inflammation of the blast injury, the bacterial load, bacterial species, and several inflammatory markers were detected. RESULTS: Compared with gauze dressing treatments, RNPT reduced bacterial load more efficiently, initiated granulation tissue formation earlier, and increased the inflammation faster. Negative pressures ranging from -10 to -25 kPa applied on the RNPT group showed beneficial effects in treating the infected soft tissue blast injury. RNPT did not significantly change both the aerobic and anaerobic bacterial composition compared with those of the gauze dressing group. CONCLUSIONS: RNPT clearly shows beneficial effects in treating the infected soft tissue blast injury in comparison with the gauze dressing therapy in swine.

New swine model of infected soft tissue blast injury.

BACKGROUND: War injuries, especially blast injuries, have a high risk of infection. However, no animal models of infected war injuries have been built in large animals, which retards both the understanding and the treatment optimization of infected war injuries. METHODS: Soft tissue blast injuries were created by explosion of electric detonators in white domestic pigs. The ultra structure of the tissue around the wound was determined by transmission electron microscope. To develop infection of blast injury wounds, the pigs were housed in a standard animal house which was disinfected periodically, and the wounds were left untreated for 3 days. Wound specimens were collected daily to determine the bacterial load and bacterial components. To determine whether infection induces tissue necrosis in infected soft tissue blast injury wounds, uninfected blast injury wounds were created as controls of infected wounds by surgical debridement daily, and the wound area and wound depth of both wounds were measured. RESULTS: The wound area and the wound depth of the soft tissue blast injury created in this study fell in the range of human moderate soft tissue war injuries, and the ultra structure of the wounds was comparable with that of human blast injury wounds. The bacterial load of uninfected wounds was under 10 colony forming unit/g during the first 3 days of injury, while that of infected wounds was over 10 colony forming unit/g after 2 days of injury. The infected soft tissue blast injury wounds contained most of the bacteria frequently isolated in battlefield wounds. In addition, infection induced evident tissue necrosis in infected blast injury wounds. CONCLUSION: The infected soft tissue blast injury wounds mimic those in human, and they can be used to address key points of treatment optimization.

Natural mineralized scaffolds promote the dentinogenic potential of dental pulp stem cells via the mitogen-activated protein kinase signaling pathway.

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.