Linagliptin's impact on lymphatic barrier and lymphangiogenesis in oral cancer with high glucose.

OBJECTIVES: Uncertainties remain regarding the effect of elevated glucose levels on lymphatic metastasis of cancer cells. Our study elucidated the mechanisms linking high glucose to lymphangiogenesis and lymphatic barrier-related factors and investigated the protective role of linagliptin against lymphatic barrier dysfunction. MATERIALS AND METHODS: A CAL-27-LEC co-culture system was established. Sodium fluorescein permeability assay observed lymphatic endothelial cell permeability. Western blotting and RT-qPCR detected protein and mRNA expression under different conditions, respectively. CCK-8, scratch wound healing, and transwell assays revealed cell migration and proliferation. Tube formation experiment tested capacity for endothelial tube formation. Immunohistochemical staining analyzed tissue sections from 43 oral cancer individuals with/without diabetes. RESULTS: In high-glucose co-culture system, we observed increased lymphatic barrier permeability and decreased expression of ZO-1 and occludin, two tight-junction proteins; conversely, the expression of PAR2, a high permeability-related protein, was increased. Following linagliptin treatment, the expression levels of VEGF-C, VEGFR-3, and PAR2 decreased, while those of ZO-1 and occludin increased. Considerably higher levels of LYVE-1 expression in individuals with diabetes than in those without diabetes. CONCLUSIONS: By ameliorating the high glucose-induced disruption of the lymphatic endothelial barrier, linagliptin may reduce lymphangiogenesis and exhibit an inhibitory effect on lymphatic metastasis in oral cancer patients with diabetes.

Downregulation of VAP-1 in OSCC suppresses tumor growth and metastasis via NF-κB/IL-8 signaling and reduces neutrophil infiltration.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is believed to play a role in inflammation. Studies have suggested that VAP-1-mediated activation of inflammation is dependent on NF-κB, leading to secretion of the interleukin (IL)-8; however, no reports have addressed the association between VAP-1 and NF-κB/IL-8 signaling in oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of VAP-1 in OSCC and further explore whether VAP-1 is involved in the regulation of neutrophil infiltration in the tumor microenvironment (TME). METHODS: Immunochemistry staining was used to observe VAP-1 expression. CCK-8 and Transwell assays were used to measure cell proliferation, migration, and invasion. OSCC xenograft mouse models were used for in vivo verification of the VAP-1 function. The expression of NF-κB and IL-8 were determined by qRT-PCR and western blot. ELISA for IL-8 was also conducted. The relationship between VAP-1 expression and neutrophil infiltration was analyzed by immunofluorescence. RESULTS: VAP-1 was overexpressed in human OSCC tissues. Downregulation of VAP-1 suppressed OSCC cells proliferation, migration, and invasion in vitro and inhibited tumor proliferation and metastasis in vivo. Additionally, downregulation of VAP-1 inhibited NF-κB/IL-8 signaling in vitro and in vivo. VAP-1 expression was positively correlated with neutrophil infiltration in human OSCC tissues. Moreover, blocking VAP-1 decreased neutrophil infiltration by reducing IL-8 production. CONCLUSIONS: VAP-1 downregulation in OSCC suppresses tumor growth and metastasis by inhibiting NF-κB/IL-8 signaling and reducing neutrophil infiltration in the TME, suggesting that VAP-1 may be a potential therapeutic target for OSCC.

Association between condylar position changes and functional outcomes after condylar reconstruction by free fibular flap.

OBJECTIVES: Stable and appropriate condyle positioning is necessary for maintaining temporomandibular joint function. It is unclear if this position remains stable in patients after free fibular flap (FFF) condylar reconstruction. We investigated whether condylar position deviated after reconstruction, and whether this affected functional recovery. MATERIALS AND METHODS: We retrospectively reviewed 43 patients who underwent conventional FFF condylar reconstruction, and 5 patients who underwent reconstruction by computer-assisted three-dimensional (3D) printing methods. Three-dimensional models were built from cone-beam computed tomography images obtained immediately postoperatively and 1-year postoperatively. The glenoid fossa and fibular condyle centers were used to measure the fibular condyle position in the models. Clinical examination indices, including maximum mouth opening (MMO), pain during chewing/mouth opening, and patient satisfaction with mastication and 1-year outcomes were assessed. RESULTS: Fibular condyle position changed significantly over 1 year in both groups (P < 0.05). Clinical examination at 1 year after the surgery showed that in the conventional group, the MMO range was ≥ 35 mm in 76.7% of patients and < 35 mm in 23.3% of patients; 4.7% experienced pain during chewing/mouth opening, and 7% were dissatisfied with treatment outcomes. In the 3D printing group, all patients had an MMO range exceeding 35 mm, none had pain, and all were satisfied with functional outcomes. CONCLUSIONS: The position of the fibular condyle deviates after reconstructive surgery, but it is unlikely to affect functional recovery. CLINICAL RELEVANCE: These findings can form the basis for evaluation of functional outcomes of patients who have previously undergone condylar reconstruction by FFF.

TGF-ß1 and IL-17A comediate the protumor phenotype of neutrophils to regulate the epithelial-mesenchymal transition in oral squamous cell carcinoma.

BACKGROUND: The role of neutrophils in cancer has been the subject of intense research in recent years. One major theme that has emerged is that not all neutrophils are equal in the field of cancer. However, it remains unclear what induces the protumorigenic or antitumorigenic phenotype predominate in tumor. Therefore, this study aimed to investigate what factors induce which of these two phenotypes of neutrophil predominate in OSCC and to explore the role of neutrophil polarization on tumor. METHODS: Immunofluorescence and immunohistochemistry staining were used to observe neutrophil infiltration and the expression of TGF-ß1 and IL-17A in OSCC tissues. Recombinant human TGF-ß1 and IL-17A were used to modulate neutrophil polarization. OSCC cell (SCC9 and SAS cell lines) migration, proliferation, invasion, stemness, and EMT were analyzed after treatment with conditioned medium from TGF-ß1/IL-17A-activated neutrophils. The levels of neutrophil-associated markers in OSCC tissues and peripheral blood were examined by immunofluorescence staining and quantitative PCR. RESULTS: Our data showed neutrophil infiltration and elevated expression of TGF-ß1 and IL-17A in OSCC tissues. The cooperative effect of TGF-ß1 and IL-17A promoted neutrophils to take on a protumor phenotype in vitro. TGF-ß1/IL-17A-activated neutrophils remarkably induced cell migration, proliferation, invasion, stemness, and EMT in OSCC cells. Additionally, OSCC patients showed increased expression of MMP9 and decreased expression of CCL3 in circulating neutrophils. CONCLUSION: TGF-ß1 and IL-17A cooperated to augment the protumor functions of neutrophils, thereby promoting the progression of OSCC cells. In addition, the combination of neutrophil-associated markers may serve as a predictive method to screen for patients with OSCC.

The association between the socioeconomic status and systemic comorbidities in patients with oral cancers: a retrospective study in Guangxi Province.

BACKGROUND: It is unclear whether and how the prevalence of systemic comorbidities in oral cancer patients would change with socioeconomic development. MATERIALS AND METHODS: A retrospective study of association between socioeconomy and prevalence of systemic comorbidities in oral cancer patients from 2003 to 2017 was performed in Guangxi Province, a southwestern part of China. According to the Union for International Cancer Control (UICC) classification, 2814 patients with squamous cell carcinoma (SCC) of the lip, oral cavity, and oropharynx and 423 patients with ameloblastoma were collected and assigned to the oral cancer group and control group, respectively. Then, comparisons between the socioeconomy and healthcare expenditure in Guangxi Province, the whole China, and the USA were carried out. RESULTS: The prevalence of systemic comorbidities in oral cancer patients increased from 0.820% in 2003 to 32.302% in 2017, which was significantly higher than that in non-cancer patients(P < 0.001) and was positively correlated with the increase in gross regional product (GRP) (r = 0.911, P < 0.001) and per capita GRP (r = 0.910, P < 0.001) of Guangxi Province. In addition, the prevalence of cardiovascular diseases has the largest correlation coefficient with GRP(r = 0.957, P < 0.001) and per capita GRP(r = 0.959, P < 0.001). And the prevalence of endocrine diseases increased by 13.402% and exhibited the most significant increase in 15 years. The per capita health care expenditure of Guangxi Province and whole China was nearly equal (P = 0.353). Although the health care expenditure of Guangxi Province had been increasing year by year, its proportion in GRP remains far below that of the USA. CONCLUSIONS: With socioeconomic growth, oral cancer patients in Guangxi Province are more common to comorbid with systemic diseases. Cardiovascular and endocrine diseases may be the most susceptible systemic comorbidities in oral cancer patients to the socioeconomic status. In order to control the prevalence of systemic diseases, the government of Guangxi Province may need to expend more budgets in the health care. CLINICAL RELEVANCE: Clinicians need to pay more attention to the detection of systemic comorbidities and the concept of multidisciplinary collaboration. Instructing oral cancer patients to treat and control systemic comorbidities is also an indispensable part in the treatment of oral cancer.

Downregulation of FOXP3 in neutrophils by IL-8 promotes the progression of oral squamous cell carcinoma.

The aim of the present study was to investigate the effects of the transcription factor forkhead box P3 (FOXP3) in neutrophils on the progression of oral squamous cell carcinoma (OSCC). Cancer tissue samples and paracarcinoma tissues were collected from 23 patients with OSCC for the current study. In addition, SCC-9, a human tongue carcinoma cell line, was co-cultured with primary human neutrophils and treated with recombinant interleukin 8 (IL-8). The effect of FOXP3 on the proliferation of SCC-9 cells was analyzed using a Cell Counting Kit 8 assay. FOXP3 expression in neutrophils was analyzed by quantitative PCR following IL-8 treatment. FOXP3 protein expression in neutrophils and the amount of IL-8 protein in the OSCC tumor microenvironment were determined by immunofluorescence analysis. The present study demonstrated that IL-8 downregulated FOXP3 mRNA expression in neutrophils. Neutrophils and peptide P60, a specific inhibitor of FOXP3, increased proliferation of SCC-9 cells. In patients with OSCC, FOXP3 protein expression in neutrophils of the stage IV group was significantly lower compared with that of the stage II and stage III groups, while IL-8 protein expression was higher in cancer tissues compared with that in paracarcinoma tissues. In summary, IL-8 in the tumor microenvironment may recruit neutrophils, and downregulation of FOXP3 in neutrophils by IL-8 may promote the progression of OSCC.

Targeting EphA4 abrogates intrinsic resistance to chemotherapy in well-differentiated cervical cancer cell line.

Alkylating reagent chemotherapy for human cancers is not curative, and relapse occurs due to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD cells after chemotherapy, a phenomenon referred to as intrinsic resistance, depends on reactive oxygen species. Well-differentiated regions of the tumor are intrinsically resistant to chemotherapy. Receptor tyrosine kinase erythropoietin-producing human hepatocellular receptor A4 (EphA4) protein is highly expressed in the well-differentiated tumor-derived cervical cancer cell line Caski, but not in poorly differentiated tumor-derived cervical cancer cell lines such as HeLa or SiHa. Here, we report that reactive oxygen species produced by cisplatin exposure induce tyrosine phosphorylation of EphA4. After observing that EphA4 is activated by cisplatin, we rationalized a combination chemotherapy that induces well-differentiated cervical cancer death. Pharmacological inhibition of EphA4 increased cisplatin-induced cell death in Caski cells. Moreover, we observed increased expression levels of the senescence marker cyclin-dependent kinase inhibitor 2A (p16) in the absence of EphA4 kinase function after stimulation of Caski cells with cisplatin exposure. Mechanistically, cisplatin induces chemotherapy resistance of Caski cells by upregulating Lyn, a Src family kinase (SFK) that interacts with EphA4, through a pathway involving reactive oxygen species. Thus, the reactive oxygen species-SFK-EphA4 axis presents new potential drug targets for chemotherapy resistance.

Comparison of the expression of TGF-ß1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α in oral squamous cell carcinoma and lymph node metastases of humans and mice.

The aim of the present study was to prove that a mouse model closely simulates human oral cancer progression by comparing the expression levels of transforming growth factor (TGF)-ß1, E-cadherin, N-cadherin, tumor protein (TP)53, RB1 inducible coiled-coil (RB1CC)1 and hypoxia inducible factor (HIF)-1α at different stages of oral squamous cell carcinoma (OSCC) in humans and mice. The expression levels of TGF-ß1, E-cadherin, N-cadherin, TP53, RB1CC1, and HIF-1α were detected by immunohistochemical staining in normal oral mucosa, oral mucosa dysplasia, OSCC primary tumor and carcinoma tissues from lymph node metastases. Tissue samples were obtained from human specimens and the Balb/c mouse model of lymphatic metastases oral carcinoma, induced by 4-nitroquinoline-1-oxide in drinking water. The results indicated no significant differences in the expression levels of TGF-ß1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α between humans and mice, at any stage of OSCC examined (P>0.05). The expression of TGF-ß1, N-cadherin, TP53 and RB1CC1 increased in different stages of OSCC in both humans and mice. The expression of E-cadherin decreased from normal oral mucosa to OSCC, and increased in lymph node metastases in both human and mouse samples. The expression of HIF-1α increased from normal oral mucosa to OSCC, and decreased in lymph node metastases in both human and mouse samples. Additionally, the expression of p53 was positively correlated with that of RB1CC1 in human and mouse samples (r=0.971, P=0.029; r=0.97, P=0.03). Overall, the similar expression of multiple molecules in both human and mouse carcinoma prove that the mouse model of lymphatic metastases from oral carcinoma established in the present study may closely mimic human oral cancer.

Coexpression of growth differentiation factor 11 and reactive oxygen species in metastatic oral cancer and its role in inducing the epithelial to mesenchymal transition.

OBJECTIVES: The aim of this study was to investigate growth differentiation factor 11 (GDF11) and reactive oxygen species (ROS) expression in metastatic oral cancer and explored their roles in inducing epithelial to mesenchymal transition (EMT). STUDY DESIGN: The expression of GDF11, ROS, and EMT-related markers was evaluated in primary tumor tissues from patients with oral squamous cell carcinoma (OSCC). SCC-9 cells, a human tongue carcinoma cell line, were treated with recombinant GDF11. Induction of EMT, expression of EMT-related markers, and the effect of ROS on EMT in SCC-9 cells were analyzed. RESULTS: Overexpression of GDF11 and ROS was observed in patients with metastatic oral cancer. Downregulated expression of E-cadherin and upregulated expression of vimentin, δ-EF1, SIP-1, MMP-2, and MMP-9 were observed in patients with metastatic oral cancer, relative to the expression of these factors in patients with nonmetastatic oral cancer. With recombinant GDF11 treatment, obvious spindle-shaped cells appeared, and gene expressions of EMT-related markers were altered in SCC-9 cells. Treatment with the powerful antioxidant N-acetylcysteine inhibited GDF11-induced EMT and cell migration. CONCLUSIONS: GDF11 induces EMT and cell migration with ROS involvement in SCC-9 cells. Overexpression of GDF11 and ROS is associated with metastatic oral cancer. GDF11 and ROS may participate in metastasis of oral cancer through EMT.

Improving Surgeons' Comfort With Prismatic Glasses During Cleft Palate Surgery: Preliminary Findings.

PURPOSE: We aimed to observe the effect of prismatic glasses on improving surgeons' comfort during cleft palate surgery. MATERIALS AND METHODS: A within-subjects design was used. We included 3 oral-maxillofacial surgeons and 6 patients with complete cleft palate in the study. One group of cleft palate patients (3 complete cleft palates) was allocated to each of the 3 surgeons not wearing prismatic glasses, and another similar group of cleft palate patients was allocated to the same 3 surgeons wearing prismatic glasses. The push-back method was performed in all cleft palate patients by all surgeons. The degree of neck flexion exhibited by all surgeons was digitally video recorded. Screen-capture images of the video recordings were collected, and neck flexion in all video recordings was analyzed. All surgeons completed a questionnaire based on a visual analog scale to assess their discomfort symptoms of the neck, shoulders, and back. Operative time and bleeding volume were recorded to assess operational efficiency. RESULTS: Use of prismatic glasses significantly reduced surgeons' working time spent in pronounced neck flexion during cleft palate surgery (P < .05), and there was a statistically significant reduction in the visual analog scale discomfort scores for the neck, back, and shoulders with the use of prismatic glasses (P < .05). However, no significant difference was found in operational time (P = .337) and bleeding volume (P = .183) attributable to the presence or absence of prismatic glasses. CONCLUSIONS: An ergonomic approach to cleft palate surgery in which surgeons wore prismatic glasses improved neck, back, and shoulder comfort.

Nonvascularized Free Transport Distraction Osteogenesis to Reconstruct the Mandibular Defect of a Patient With an Ossifying Fibroma: A Case Report.

We report on a case in which trifocal distraction osteogenesis was applied in a patient with a mandibular defect caused by resection of an ossifying fibroma. During the surgical procedure, we accidentally dissected the surrounding periosteal tissue of the left transport completely in the process of transport disc preparation and made the disc into a free bone graft. However, we still used this transport and successfully completed the distraction osteogenesis. The patient's facial and occlusal function were regained after treatment.

PDGF α receptor is a mediator for Cisplatin-induced Met expression.

For decades, platinum drugs have been the mainstay of cancer treatment. However, over time, drug resistance develops, leaving few treatment options. Here we show that platelet-derived growth factor α receptor (PDGF α receptor)-mediated signaling plays a key role in hepatocyte growth factor (HGF) receptor (c-Met) upregulation, which in turn is thought to play an important role in chemotherapy resistance. PDGF α receptor inhibition eliminates cisplatin-dependent Met expression in cervical cancer cell lines. PDGF α receptor inhibitors are widely used in clinical settings, suggesting that the clinical translation of our findings could reduce the suffering of people from drug resistance.

Development of a 4-nitroquinoline-1-oxide model of lymph node metastasis in oral squamous cell carcinoma.

OBJECTIVES: Lymph node metastasis is one of the most important prognostic factors in oral squamous cell carcinoma. However, regional lymph node metastasis from oral cancer has barely been investigated in 4-nitroquinoline-1-oxide (4NQO) models. In this study, we developed a 4NQO-treated mouse model of oral squamous cell carcinoma that displayed lymph node metastasis and examined the occurrence and timing of the lymph node metastasis. MATERIALS AND METHODS: BALB/c mice were treated with 200 µg/ml 4NQO in drinking water for 20 weeks and then followed for 4 weeks. Four mice were sacrificed bi-weekly, and oral lesion and regional lymph node metastasis were assessed during the week 25-32 and week 33-40 observation periods. RESULTS: Tumor formation and local lymph enlargement were observed from 25 weeks onwards. The presence of metastatic tumor cells in the enlarged lymph nodes was confirmed via the detection of tumor infiltration by HE staining and cytokeratin positivity by immunostaining at both 28 and 36 weeks. The incidence rates of oral squamous cell carcinoma and local lymph node metastasis were 68.8% and 12.5% in the week 25-32 observation period, and 100% and 100% in the week 33-40 observation period, respectively. CONCLUSIONS: A lymph node metastasis model involving mice being subjected to high-dose 4NQO treatment for a long period and then followed for a prolonged period was established. This model might provide a platform for studying the mechanism of lymphatic metastasis in oral carcinoma, leading to the development of new therapies for advanced oral carcinoma.

HPV16E6-dependent c-fos expression contributes to AP-1 complex formation in SiHa cells.

To date, the major role of HPV16E6 in cancer has been considered to be its ability to inhibit the p53 tumor-suppressor protein, thereby thwarting p53-mediated cytotoxic responses to cellular stress signals. Here, we show that HPV16E6-dependent c-fos oncogenic protein expression contributes to AP-1 complex formation under oxidative stress in SiHa cells (HPV16-positive squamous cell carcinoma of the cervix). In addition, we examined the role of HPV16E6 in TGF-α-induced c-fos expression and found that the c-fos protein expression induced by TGF-α is HPV16E6 dependent. Thus, our results provide the first evidence that HPV16E6 contributes to AP-1 complex formation after both ligand-dependent and independent EGFR activation, suggesting a new therapeutic approach to the treatment of HPV-associated tumors.

[Expression and significance of nitric oxide synthase in mandibular distraction osteogenesis of canine].

OBJECTIVE: To investigate the expression and significance of nitric oxide synthase (NOS) during mandibular distraction osteogenesis and bone trauma healing, to futher study the mechanism of distraction osteogenesis. METHODS: Twenty-eight adult dogs were randomly divided into DO group (12 dogs), acutely lengthening group (12 dogs) and control group (4 dogs). Immunohistochemical examination were carried out to test the expression of NOS during the sixth day in distraction period, the second and eighth week of consolidation. RESULTS: In DO group infiltration of inflammatory cell was found in the distraction gap, more red blood cells (RBC) leak out around vascellum and matrix in the sixth day in distraction period. The expression of local iNOS (inducible NOS) and eNOS (endothelinal NOS) in DO gruoup and acutely lengthening group was higher than that in the control group (P < 0.05), the expression of local iNOS and eNOS in acutely lengthening group was lower than that in DO group (P < 0.05) in the early of distraction period and the consolidation. The expression of local iNOS and eNOS was no statistic difference between three groups (P > 0.05) in the eighth week of consolidation. CONCLUSION: NOS as a sensitive index of tissue trauma are highly expressed, and RBC was found leaking out in the early of distraction, indicating that micro-trauma to some extent may occurr during DO procedure, the micro-trauma may be one of the significant factors which increasing regeneration of bone during DO.

Regulation of chemokine production via oxidative pathway in HeLa cells.

Inflammation is associated with disease progression and, by largely unknown mechanisms, has been said to drive oncogenesis. At inflamed sites, neutrophils deploy a potent antimicrobial arsenal that includes proteinases, antimicrobial peptides, and ROS. Reactive oxygen species (ROSs) induce chemokines. In the present study, the concentrations of IL-8 in culture supernatants of HeLa cells treated with ROS were determined by enzyme-linked immunosorbent assay. We used o-phenanthroline to deplete Fe(2+) in order to investigate the mechanisms through which ROSs induce IL-8 secretion in our system. The iron chelator o-phenanthroline effectively inhibited H(2)O(2)-induced ERK2 activation. Enzyme-linked immunosorbent assays showed that IL-8 protein secretion was elevated in ROS-treated HeLa cells. When Fe(2+) was removed from these cells, IL-8 secretion was inhibited. Collectively, these results indicate that Fe(2+)-mediated Erk pathway activation is an important signal transduction pathway in ROS-induced IL-8 secretion in epithelial cells.

[Cloning of human bone morphogenetic protein-2 gene and the construction of its eukaryotic expression vector].

OBJECTIVE: To clone human bone morphogenetic protein-2 (hBMP2) gene and construct its eukaryotic expression vector pcDNA3.1 -hBMP2. METHODS: Human BMP2 gene was amplified by RT-PCR method from human osteosarcoma cells and constructed into eukaryotic expression vector pcDNA3.1-hBMP2. The gene in the vector pcDNA3.1-hBMP2 was identified by PCR amplification, enzyme digestion and DNA sequencing. RESULTS: The cloned DNA was confirmed to be hBMP-2 gene. CONCLUSION: In this study, hBMP2 gene is successfully cloned and its eukaryotic expression vector pcDNA3.1-hBMP2 is constructed, which provides the foundation of using BMP2 gene therapy to accelerate new bone formation in distraction osteogenesis.

[The effect of dog bilateral distraction osteogensis in temporomandibular joint].

OBJECTIVE: To investigate the effect of bilateral mandibular distraction osteogenesis in the condyles. METHODS: 16 adult hybrid dogs were randomly divided into normal control group and experiment group. Experimental dogs underwent bilateral mandibular osteodistraction at a rate of 1 min/day. 4 dogs were killed respectively in distraction period, 2 and 8 weeks after completion of 10 days distraction. The bilateral condyles specimens were harvested and examined with histological and immunohistochemical methods. RESULTS: Compared with normal control group, various degrees of irregularities and erosion were found in fibrocartilage of condyle in experiment group, including damage in fibrous layer, hyperplasia layer and proliferative layer and osteogenic activity in cartilage layer. A significant increase of TGF-beta1 expression was also found in experiment groups. TGF-beta1 positive staining was noted in hypertrophic cell, matrix and chondroblast, osteoblast and matrix in osteogenic activity areas. These changes were the most obvious in 2 weeks after completion of distraction. CONCLUSION: Gradual bilateral mandibular distraction at a rate of 1 mm/day brought degenerative changes of condyle, but the changes are reversible.