Arecoline Induces ROS Accumulation, Transcription of Proinflammatory Factors, and Expression of KRT6 in Oral Epithelial Cells.

Areca nut is a major contributor to the high prevalence of oral cancer in Asia. The precise mechanisms by which areca nut stimulates mucosal cells and contributes to the progression of oral cancer urgently require clarification. The current study aimed to assess the effects of arecoline on the normal human gingival epithelium cell line S-G. Cell viability, levels of reactive oxygen species (ROS), protein expression, cellular morphology, and gene expression were evaluated using the MTT test, flow cytometry, Western blot analysis, optical or confocal microscopy, and RT-qPCR. Keratin (KRT6) analysis involved matched normal and cancer tissues from clinical head and neck specimens. The results demonstrated that 12.5 µg/mL of arecoline induced ROS production, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNA expression in S-G cells. This activation of the MAPK/ERK pathway increased KRT6 expression while limiting cell migration. In head and neck cancer tissues, KRT6B gene expression exceeded that of normal tissues. This study confirms that arecoline induces ROS accumulation in normal cells, leading to the secretion of proinflammatory factors and KRT6 expression. This impedes oral mucosal healing, thereby promoting the progression of oral cancer.

Identification of the perpetrator imperatorin in Xin-yi-san-theophylline interaction: observed and predicted herb-drug interaction in rats.

OBJECTIVES: Theophylline is a bronchodilator with a narrow therapeutic index and primarily metabolised by cytochrome P450 (CYP) 1A2. Xin-yi-san (XYS) is a herbal formula frequently used to ameliorate nasal inflammation. This study aimed to investigate the effects of XYS and its ingredient, imperatorin, on theophylline pharmacokinetics in rats. METHODS: The kinetics of XYS- and imperatorin-mediated inhibition of theophylline oxidation were determined. Pharmacokinetics of theophylline were analysed. Comparisons were made with the CYP1A2 inhibitor, fluvoxamine. KEY FINDINGS: XYS extract and its ingredient, imperatorin, non-competitively inhibited theophylline oxidation. Fluvoxamine (50 and 100 mg/kg) and XYS (0.5 and 0.9 g/kg) significantly prolonged the time to reach the maximum plasma concentration (tmax) of theophylline by 3-10 fold. In a dose-dependent manner, XYS and imperatorin (0.1-10 mg/kg) treatments significantly decreased theophylline clearance by 27-33% and 19-56%, respectively. XYS (0.9 g/kg) and imperatorin (10 mg/kg) significantly prolonged theophylline elimination half-life by 29% and 142%, respectively. Compared with the increase (51-112%) in the area under curve (AUC) of theophylline by fluvoxamine, the increase (27-57%) by XYS was moderate. CONCLUSIONS: XYS decreased theophylline clearance primarily through imperatorin-suppressed theophylline oxidation. Further human studies are essential for the dose adjustment in the co-medication regimen.

Potential to Eradicate Cancer Stemness by Targeting Cell Surface GRP78.

Cancer stemness is proposed to be the main cause of metastasis and tumor relapse after conventional therapy due to the main properties of cancer stem cells. These include unlimited self-renewal, the low percentage in a cell population, asymmetric/symmetric cell division, and the hypothetical different nature for absorbing external substances. As the mechanism of how cancer stemness is maintained remains unknown, further investigation into the basic features of cancer stemness is required. Many articles demonstrated that glucose-regulated protein 78 (GRP78) plays a key role in cancer stemness, suggesting that this molecule is feasible for targeting cancer stem cells. This review summarizes the history of finding cancer stem cells, as well as the functions of GRP78 in cancer stemness, for discussing the possibility of targeting GRP78 to eradicate cancer stemness.

Nitrogen-doped carbon nanodots prepared from polyethylenimine for fluorometric determination of salivary uric acid.

Stable and low-cost carbon dots (C-dots) were prepared from polyethylenimine (PEI) by a hydrothermal method. It is found that the fluorescence of the C-dots (best measured at excitation/emission wavelengths of 365/473 nm) is quenched by selective oxidation of surface PEI by periodate but recovers in the presence of uric acid (UA). It is assumed that this is due to the selective reduction of the nitrone groups to hydroxylamine groups by UA. The findings were used to design a fluorometric method for determination of UA that has a 2.3 nM detection limit. This is lower than that of reported fluorometric and enzymatic assays. The performance of the method has been validated by determination of UA in samples of human saliva. It is found that the results agree well with those obtained by a commercial UA assay. Graphical abstract Schematic presentation of the polyethylenimine (PEI) carbon nanodots (C-dots) as a fluorescent probe for uric acid. Their fluorescence is quenched by periodate (IO4-) due to oxidative formation of nitrone groups, an subsequently restored due to reduction by uric acid (UA).

Author Correction: Rapid Staining of Circulating Tumor Cells in Three-Dimensional Microwell Dialysis (3D-µDialysis) Chip.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

Rapid Staining of Circulating Tumor Cells in Three-Dimensional Microwell Dialysis (3D-µDialysis) Chip.

The conventional techniques to detect circulating tumour cells (CTCs) are lengthy and the use of centrifugal forces in this technique may cause cell mortality. As the number of CTCs in patients is quite low, the present study aims towards a gentler diagnostic procedure so as not to lose too many CTCs during the sample preparation process. Hence, a Three-Dimensional Microwell dialysis (3D-µDialysis) chip was designed in this study to perform gentle fluorescence-removal process by using dialysis-type flow processes without centrifuging. This leads to a minimum manual handling of CTCs obtained in our study without any contamination. In addition, a rapid staining process which necessitates only about half the time of conventional techniques (35 minutes instead of 90 minutes) is being illustrated by the employment of dialysis process (by dynamically removing water and waste at once) instead of only static diffusion (by statically removing only waste by diffusion). Staining efficiency of our technique is improved over conventional staining because of the flow rate in 3D-µDialysis staining. Moreover, the staining process has been validated with clinical whole blood samples from three TNM stage IV colon cancer patients. The current technique may be termed as "miniature rapid staining and dialysing system".

MiR-520b as a novel molecular target for suppressing stemness phenotype of head-neck cancer by inhibiting CD44.

Cancer stem cells preferentially acquire the specific characteristics of stress tolerance and high mobility, allowing them to progress to a therapy-refractive state. To identify a critical molecule to regulate cancer stemness is indispensable to erratically cure cancer. In this study, we identified miR-520b as a novel molecular target to suppress head-neck cancer (HNC) with stemness phenotype. MiR-520b inhibited cellular migration and invasion via the mechanism of epithelial-mesenchymal transition. It also sensitized cells to therapeutic drug and irradiation. Significantly, miR-520b suppressed spheroid cell formation, as well as reduced expressions of multiple stemness regulators (Nestin, Twist, Nanog, Oct4). The CD44 molecule was identified as a direct target of miR-520b, as shown by the reverse correlative expressions, the response to miR-520 modulation, the luciferase reporter assay, and the functional rescue analyses. These cellular results were confirmed by a tumor xenograft mice study. Administration of miR-520b dramatically restrained tumorigenesis and liver colonization. Conversely, miR-520b silencing led to an acceleration of tumor growth. Taken together, our study demonstrated that miR-520b inhibits the malignancy of HNC through regulation of cancer stemness conversion by targeting CD44. MiR-520b may serve as an emerging therapeutic target that may be further developed for the intervention of refractory HNC.

GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway.

Radiotherapy is an integral part for the treatment of head and neck cancer (HNC), while radioresistance is a major cause leads to treatment failure. GDF15, a member of the TGF-ß superfamily, is hypothesized to participate in various types of homeostasis. However, the potential role of this molecule in regulation of radiosensitivity remains unclear. In this study, we demonstrated that GDF15 contributed to radioresistance of HNC, as determined by both gain- and lost-of-functional experiments. These results were achieved by the induction of mitochondrial membrane potential and suppression of intracellular reactive oxygen species (ROS). We further showed that GDF15 facilitated the conversion of cancer stemness, as assessed by the promotion of CD44+ and ALDH1+ cell populations and spheroid cell formation. At molecular level, GDF15 conferred to these cellular functions was through phosphorylated SMAD1 proteins to elite downstream signaling molecules. These cellular results were further confirmed in a tumor xenograft mouse study. Taken together, our results demonstrated that GDF15 contributed to radioresistance and cancer stemness by regulating cellular ROS levels via a SMAD-associated signaling pathway. GDF15 may serve as a prediction marker of radioresistance and a therapeutic target for the development of radio-sensitizing agents for the treatment of refractory HNC.

Vascular endothelial growth factor-A stimulates Snail expression in breast tumor cells: implications for tumor progression.

The E-cadherin transcriptional repressor Snail is a prognostic marker for metastatic breast carcinoma, as well as a critical determinant of tumor growth and recurrence. We define a non-angiogenic, autocrine function for the vascular endothelial growth factor-A (VEGF-A) in regulating Snail expression in breast tumor cells. The transfection of well-differentiated breast tumor cells with VEGF-A increases Snail mRNA and protein levels, resulting in reduced E-cadherin expression. Conversely, reducing endogenous VEGF-A expression in poorly differentiated breast tumor cells by siRNA transfection decreases Snail levels. Our studies demonstrate that VEGF and the VEGF receptor Neuropilin-1 increase Snail expression by suppressing the Glycogen Synthase Kinase-3 (GSK-3), an established inhibitor of Snail transcription and protein stability. The VEGF-A neutralizing antibody Avastin was recently approved by the FDA for the treatment of metastatic breast cancer. We present the provocative finding that beyond its anti-angiogenic activity, Avastin can reduce Snail expression in breast tumor cells. Collectively, this work describes a novel autocrine function for VEGF in breast tumor cells in driving the expression of Snail, a breast tumor progression factor. Based on our demonstration that Avastin reduces Snail expression in breast tumor cells, we propose that the treatment of early stage breast cancer patients with Avastin may impede tumor progression.

Raffinee in the treatment of spinal cord injury: an open-labeled clinical trial.

The purpose of this preliminary clinical trial was to assess the safety and therapeutic trend of Raffinee, a mixture of free radical scavengers derived from natural products, on subacute spinal cord injury (SCI). SCI patients (three females and seven males) were enrolled from three medical centers in Taiwan. Their ages ranged from 18 to 76 years, with an average of 47.6 +/- 19.0 years. There were four incomplete and six complete cases in this study. Standard regimens for SCI at acute stage were given at each hospital. Approximately 7 weeks (35.4 +/- 13.9 days) after SCI, 2 U of Raffinee was given orally before meals three times a day for 6 weeks. Patients received motor, sensory, and activities of daily living (ADL) assessments before and after the treatment with Raffinee every week for 6 weeks. The Wilcoxon signed-ranks test was used for statistical analysis. Significant motor and sensory recovery began in the second week of Raffinee treatment, and significant ADL recovery was also noted in the third week. Functional recovery was more prominent in incomplete cases. Raffinee appeared to be safe in the subacute stage of SCI and may be an effective adjuvant therapy for enhancing functional recovery. Further clinical studies including double-blinded randomized placebo-controlled trials with follow-up for more than 1 year are necessary to validate the effectiveness of Raffinee in SCI.