Differences in the response of normal oral mucosa, oral leukoplakia, oral squamous cell carcinoma-derived mesenchymal stem cells, and epithelial cells to photodynamic therapy.

OBJECTIVE: The objective of this study is to investigate the variances in transcriptome gene expression of normal oral mucosa-derived mesenchymal stem cell (OM-MSC), oral leukoplakia-derived MSC (OLK-MSC) and oral squamous cell carcinoma-derived MSC(OSCC-MSC). as Additionally, the study aims to compare the in vitro proliferation, migration, invasion ability, and response to photodynamic therapy (PDT) of these three MSC, HOK, DOK, leuk1, and Cal27 cell lines. METHODS: HOK, DOK, leuk1, Cal27 cells were cultured in vitro. 3 MSC cells were obtained from OM, OLK, OSCC tissue (n = 3) and identified through flow cytometry. They were also cultured in vitro for osteogenic and lipogenic-induced differentiation. Based on the Illumina HiSeq high-throughput sequencing platform, OM-MSC, OLK-MSC, OSCC-MSC (n = 3) were subjected to transcriptome sequencing, functional annotation, and enrichment analysis of differentially expressed genes and related genes. CCK8 assay, wound healing assay, and transwell assay were performed to compare the proliferation, migration, and invasion of the seven types of cells. The 7 cells were incubated with 0, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM of the photosensitizer (5-aminolevulinic acid, 5-ALA) in vitro. Subsequently, they were irradiated with a 150 mM, 635 nm laser for 1 min, and the cell activity was detected using the CCK8 assay after 24 h. The mitochondrial changes in the 7 cells before and after the treatment of PDT were detected using the JC-10 probe, and the changes in ATP content were measured before and after the PDT treatment. RESULTS: OM-MSC, OLK-MSC, and OSCC-MSC expressed positive MSC surface markers. After osteogenic and lipogenic-induced differentiation culture, stained calcium nodules and lipid droplets were visible, meeting the identification criteria of MSC. Pathway enrichment analysis revealed that the differentially expressed genes (DEGs) of OSCC-MSC compared to OLK-MSC were primarily associated with the PI3K-Akt signaling pathway and tumor-related pathways. OSCC-MSC exhibited stronger migratory and invasive abilities compared to Cal27. The IC50 values required for OM, OLK, and OSCC-derived MSC were lower than those required for epithelial cells treated with PDT, which were 1.396 mM, 0.9063 mM, and 2.924 mM, respectively. Cell membrane and mitochondrial disruption were observed in seven types of cells after 24 h of PDT treatment. However, HOK, DOK, leuk1, and Cal27 cells had an ATP content increased. CONCLUSIONS: OLK, OSCC epithelial cells require higher concentrations of 5-ALA for PDT treatment than MSC of the same tissue origin. The concentration of 5-ALA required increases with increasing cell malignancy. Differences in the response of epithelial cells and MSC to PDT treatment may have varying impacts on OLK recurrence and malignancy.

Effect of ALA-PDT on inhibition of oral precancerous cell growth and its related mechanisms.

BACKROUND: Early treatment of oral precancerous lesions is considered as a key strategy for in oral carcinogenesis prevention. Increasing evidence has suggested that the transforming growth factor beta (TGF-ß) signaling pathway is tightly involved in the process of oral-carcinogenesis. In this study, we investigated the inhibition effect and potential mechanism of 5-aminolaevulinic acid photodynamic therapy (ALA-PDT) in human oral precancerous cells via TGF-ß pathway. MATERIALS AND METHODS: Here, the dysplastic oral keratinocyte (DOK) cells were incubated with ALA concentration of 1 mM/mL for 4 h and then irradiated with a Helium-Neon (He-Ne) ion laser at 633 nm (200 mW/cm2). The control cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium. We analyzed the differentially expressed genes and correlated pathways in oral precancerous cells following ALA-PDT using Affymetrix microarrays. TGF-ß pathway was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. Bioinformatics analysis was performed to evaluate the expression of TGF-ß1 in human oral cancer samples and adjacent normal samples. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), flow cytometry, 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and wound healing assay were used to assess the effects of ALA-PDT plus TGF-ß receptor inhibitor (LY2109761) in DOK cells. RESULTS: The TGF-ß signaling could exert in suppressive effects on DOK cells after ALA-PDT. The cell proliferation and migration rate of DOK cells was significantly reduced and apoptosis and ROS generation induced more effectively by ALA-PDT combined with LY2109761. Furthermore, cell cycle analysis revealed that the combined treatment resulted in G0/G1 phase arrest. CONCLUSIONS: ALA-PDT suppresses the growth of oral precancerous cells by regulating the TGF-ß signaling pathway, and its suppressive effect was enhanced using LY2109761. These results indicate that it could be a promising alternative treatment against oral precancerous lesions.

Mesenchymal stem cell-exosome-mediated matrix metalloproteinase 1 participates in oral leukoplakia and carcinogenesis by inducing angiogenesis.

BACKGROUND: In the malignant progression of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC), the density of microvessels and expression of angiogenesis-related molecules increases. Emerging evidence indicates that mesenchymal stem cells (MSCs) play an indispensable role in the tumor microenvironment. However, the role and mechanism of action of oral MSCs in inducing angiogenesis remain unclear. Therefore, it is necessary to explore the molecules and mechanisms that play a role in the tissue microenvironment. METHODS: Exosomes were collected from normal oral mucosa (N-Exo), OLK (OLK-Exo), and OSCC (Ca-Exo) MSCs, and their pro-angiogenic capacity was evaluated in human umbilical vein endothelial cells (HUVECs) and a subcutaneously implanted tumor model in nude mice. Quantitative proteomics analysis was used to compare the exosome-derived proteins between N-Exo, OLK-Exo, and Ca-Exo. RESULTS: Compared with that of the N-Exo and control, OLK-Exo and Ca-Exo treatment significantly promoted HUVEC migration, invasion, and tube-formation capability. In the nude mice model, immunofluorescence of CD31 showed that OLK-Exo and Ca-Exo substantially improved neovascularization around the grafts. Quantitative proteomics analysis revealed that matrix metalloproteinase 1 (MMP1) levels were significantly higher in the OLK-Exo and Ca-Exo groups than in the N-Exo groups. Silencing MMP1 expression reversed the functional promoting effect of OLK-Exo and Ca-Exo on HUVECs. CONCLUSION: Exosomes from OLK-MSCs and Ca-MSCs have a stronger pro-angiogenic ability through high MMP1 content. This new finding provides insight into the intervention with the secretion of MSC-derived exosomes, which may be an innovative strategy for carcinogenesis.

Automated quality assessment of chest radiographs based on deep learning and linear regression cascade algorithms.

OBJECTIVES: Develop and evaluate the performance of deep learning and linear regression cascade algorithms for automated assessment of the image layout and position of chest radiographs. METHODS: This retrospective study used 10 quantitative indices to capture subjective perceptions of radiologists regarding image layout and position of chest radiographs, including the chest edges, field of view (FOV), clavicles, rotation, scapulae, and symmetry. An automated assessment system was developed using a training dataset consisting of 1025 adult posterior-anterior chest radiographs. The evaluation steps included: (i) use of a CNN framework based on ResNet - 34 to obtain measurement parameters for quantitative indices and (ii) analysis of quantitative indices using a multiple linear regression model to obtain predicted scores for the layout and position of chest radiograph. In the testing dataset (n = 100), the performance of the automated system was evaluated using the intraclass correlation coefficient (ICC), Pearson correlation coefficient (r), mean absolute difference (MAD), and mean absolute percentage error (MAPE). RESULTS: The stepwise regression showed a statistically significant relationship between the 10 quantitative indices and subjective scores (p < 0.05). The deep learning model showed high accuracy in predicting the quantitative indices (ICC = 0.82 to 0.99, r = 0.69 to 0.99, MAD = 0.01 to 2.75). The automatic system provided assessments similar to the mean opinion scores of radiologists regarding image layout (MAPE = 3.05%) and position (MAPE = 5.72%). CONCLUSIONS: Ten quantitative indices correlated well with the subjective perceptions of radiologists regarding the image layout and position of chest radiographs. The automated system provided high performance in measuring quantitative indices and assessing image quality. KEY POINTS: • Objective and reliable assessment for image quality of chest radiographs is important for improving image quality and diagnostic accuracy. • Deep learning can be used for automated measurements of quantitative indices from chest radiographs. • Linear regression can be used for interpretation-based quality assessment of chest radiographs.

Chitosan-tripolyphosphate nanoparticles-mediated co-delivery of MTHFD1L shRNA and 5-aminolevulinic acid for combination photodynamic-gene therapy in oral cancer.

BACKGROUND: Rationally designed nanostructured materials can produce improved drug carriers that play an increasingly important role in cancer treatment. In comparison with conventional drug combination approaches, using co-delivery systems of multiple drugs achieves sophisticated targeting strategies and multifunctionality. METHODS: First, a nano-co-delivery of chitosan/tripolyphosphate (CS-TPP) was synthesized and characterized combining 5-aminolevulinic acid photodynamic therapy (ALA-PDT) with methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) shRNA. In this report, we investigated the efficacy of the simultaneous delivery of shRNA/photosensitizer on the gene expression of oral squamous cell carcinoma (OSCC) cells. The efficacy of CS-TPP-(shMTHFD1L-ALA)-PDT in inducing apoptosis and in generating of reactive oxygen species (ROS) in vitro was then assessed by Annexin V-PI and DCFH-DA assays respectively. In vivo therapeutic experiments were conducted in well-established orthotopic animal models of HNSCC. RESULTS: The results showed that the CS-TPP-(shMTHFD1L-ALA) nanoparticles (NPs) were approximately 145 nm in size. The cytotoxicity of OSCC cells was significantly increased by co-delivery of MTHFD1L shRNA and ALA-PDT compared with other groups. Furthermore, individual and combined therapies revealed remarkable pro-apoptotic, ROS and anti-tumorigenesis effects, and CS-TPP-(shMTHFD1L-ALA)-PDT had additive effects in vitro and in vivo. CONCLUSION: These observations indicate that CS-TPP-(shMTHFD1L-ALA) NPs may be an ideal candidate for gene/photosensitizer delivery.

NAD-dependent methylenetetrahydrofolate dehydrogenase inhibits oral squamous cell carcinoma cell proliferation and promotes apoptosis.

BACKGROUND: NAD-dependent methylenetetrahydrofolate dehydrogenase catalyzes the conversion of 10-formyltetrahydrofolate to formate in embryonic and adult mammalian mitochondria. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is a folate cycle enzyme that is involved in the development of various diseases including cancer. However, the specific mechanisms in oral squamous cell carcinoma (OSCC) are unclear. We analyzed the functional routes of MTHFD1L in OSCC cells. METHODS: MTHFD1L expression in OSCC was analyzed using data from The Cancer Genome Atlas (TCGA) database. Then, the levels of mRNA were measured in OSCC and para-tumor oral tissues using Affymetrix microarrays. Additionally, the effects of short hairpin RNA (shRNA)-induced MTHFD1L silencing on the biological behavior of OSCC were assessed in vitro and in vivo, and the potential molecular mechanisms underlying MTHFD1L activity were also investigated. RESULTS: A TCGA database analysis of RNA sequencing revealed that MTHFD1L levels were higher in tumor tissue than in adjacent tissues. Immunohistochemical staining and Kaplan-Meier survival analysis also indicated that MTHFD1L upregulation is associated with a poor prognosis in OSCC. The knockdown of MTHFD1L suppressed cell proliferation, colony formation, and tumorigenesis, while it induced apoptosis in OSCC. Mechanistically, a microarray analysis showed that MTHFD1L suppressed c-MYC and activated p53 signaling by regulating the protein expression of TP53, GADD45A, FAS and JUN. CONCLUSIONS: MTHFD1L may be involved in OSCC progression via the c-MYC gene and p53 signaling and may serve as a novel target and orientation for tumor therapy.

Construction of a pH-responsive, ultralow-dose triptolide nanomedicine for safe rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is a chronicautoimmune disease, marked by joint swelling and pain, articular synovial hyperplasia, as well as cartilage and bone destruction. Triptolide (TP) is an anti-inflammatory molecule but its use to treat RA is limited due to poor solubility and extremely high toxicity. In this study, by encapsulating TP into a star-shaped amphiphilic block copolymer, POSS-PCL-b-PDMAEMA, we engineered a pH-sensitive TP-loaded nanomedicine (TP@NPs) to simultaneously reduce the toxicity of TP and improve its therapeutic efficacy. TP@NPs shows a uniform spherical structure with a hydrodynamic diameter of ~92 nm and notable pH-responsiveness. In vitro TP@NPs showed reduced cytotoxicity and cell apoptosis of treated RAW264.7 cells compared to free TP. And in vivo intravenous injection of indocyanine green-labeled NPs into a collagen-induced arthritis model in mice showed that the engineered compound had potent pharmacokinetic and pharmacodynamic profiles, while exhibiting significant cartilage-protective and anti-inflammatory effects with a better efficacy and neglible systemic toxicity even at an ultralow dose compared to free TP. These results suggest that TP@NPs may be a safe and effective therapy for RA and other autoimmune diseases.

Multifactor analysis of patients with oral sensory complaints in a case-control study.

BACKGROUND: There are an increasing number of patients with oral sensory complaints (OSCs) presenting to our dental clinic. For most dentists, it is difficult to distinguish burning mouth syndrome (BMS) from other oral mucosal diseases that may cause symptoms such as burning mouth. It is beneficial to effectively distinguish OSC patients to reduce misdiagnosis and eliminate burning symptoms as much as possible. METHODS: Patients with oral burning sensations in the oral mucosal disease clinic were collected from the Peking University Hospital of Stomatology between September 1, 2014 and December 31, 2018. After excluding oral candidiasis, anemic stomatitis, dental material allergy, and other diseases from patients with oral sensory complaints, basic conditions such as gender, age, education level, job status, hyperglycemia, hypertension, hyperlipidemia, history of brain abnormalities, history of cervical spondylitis, history of thyroid disease, history of thyroid disease and insomnia were obtained. The BMS patients were compared with the control group. The t test and Chi-square test were used for statistical analysis to compare the clinical symptoms of these diseases and explore the risk factors for BMS. RESULTS: In this case-control study, 395 patients (321 females and 74 males, mean age 55.26 ±â€Š10.51 years) with oral sensory complaints and 391 healthy controls (281 females and 110 males, mean age 47.11 ±â€Š13.10 years) were enrolled, among which, 8.4% (33/395) had oral candidiasis, 1.3% (5/395) had dental material allergy, 0.8% (3/395) had anemic stomatitis and 0.5% (2/395) had lichen planus. A total of 352 patients were eventually diagnosed with BMS. Anxiety and depression were more severe in BMS patients, as were the incidences of sleep disorders and brain abnormalities. Logistic regression analysis showed that age (odds ratio [OR] = 2.79, 95% confidence interval [CI]: 1.61-4.83, P < 0.001), total cholesterol level (OR = 2.92, 95% CI: 1.32-6.50, P = 0.009) and anxiety score (OR = 1.75, 95% CI: 1.01-2.77, P = 0.017) significantly increased the incidence of BMS. Patients with hyperglycemia (OR = 0.46, 95% CI: 0.23-0.89, P = 0.022), low body mass index (BMI: OR = 0.57, 95% CI: 0.34-0.93, P = 0.026) and low education level (OR = 3.43, 95% CI: 1.91-6.15, P < 0.001) were more likely to suffer from BMS. CONCLUSIONS: Oral candidiasis, anemic stomatitis, and dental material allergy with burning symptoms should be excluded from patients with BMS. It is recommended to conduct a questionnaire survey (including anxiety and depression), blood cell analysis, and salivary fungus culture for all patients with an oral burning sensation. It is necessary to conduct a patch test on patients with oral burning sensations and metal restorations.

Differential in vitro sensitivity of oral precancerous and squamous cell carcinoma cell lines to 5-aminolevulinic acid-mediated photodynamic therapy.

OBJECTIVES: The clinical effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) may be correlated with the degree of dysplasia of cancer tissues, but much is still unknown regarding the differences in its effectiveness, especially in oral cancer and precancerous lesions. The aim of this study is to compare the effects of ALA-PDT on a human oral precancerous cell line (DOK) and an oral squamous cell carcinoma cell line (CAL-27). METHODS: First, we explored the dose- and time-dependent responses of DOK and CAL-27 cells to ALA-PDT. DOK and CAL-27 cells were incubated with various concentrations of ALA (from 0.25 to 2 mM), followed by PDT using laser irradiation at 635 nm. The resulting photocytotoxicity was assessed in both cell lines using MTT assays. Further, apoptosis was assessed using flow cytometry, reactive oxygen species (ROS) generation was evaluated with 2,7-dichlorofluorescein diacetate (DCFH2-DA), and the response to treatment was examined via RT-qPCR and Western blotting to measure the mRNA and protein expression levels of matrix metallopeptidase 2 (MMP-2) and MMP-9. RESULTS: ALA-PDT inhibited the proliferation of DOK and CAL-27 cells in a dose- and time-dependent manner. Dose-effect and inhibition-time relationships were also found. The rates of DOK and CAL-27 cell apoptosis when the ALA dose was 1 mM were 30.66 ±â€¯3.10% and 75.40 ±â€¯1.29%, respectively (P < 0.01). Following PDT, compared with DOK cells, the ROS level in CAL-27 cells was significantly increased and was correlated with an increase in the ALA concentration. Mechanistically, both the mRNA and protein expression levels of MMP-2 and MMP-9 were found to be regulated in both cell types after ALA-PDT. CONCLUSION: ALA-PDT effectively killed DOK and CAL-27 cells in a dose- and time-dependent manner in vitro. However, under the same conditions, the susceptibilities of these cell lines to ALA-PDT were different. Further studies are necessary to confirm whether this difference is present in clinical oral cancer and precancerous lesions.