Novel regimens of phytopolyphenols and celecoxib enhancing efficacy and selectivity of anticancer effects of chemotherapeutic agents on cultured cancer cells.

Background/purpose: Explorations of novel regimens enhancing efficacy and selectivity of chemotherapeutic agents are urgent to solve the problems of cancer therapy. This study aimed to explore synergistic anticancer effects of novel regimens of phytopolyphenols [curcumin (C), tea polyphenols (G) or GC] with celecoxib (Cl) and ZnSO4. Materials and methods: Antiproliferative effects of drugs on cultured cancer cells and pathogenic biofilms were assayed by MTT and optical density (OD600) respectively; their inhibition on efflux pump (Na+-K+-ATPase) was measured by colorimetric methods. Synergistic (CI < 1) anticancer effects were evaluated by the equations of combination index (CI) and efficacy index (EI). Results: Both Cl and methotrexate (MTX) alone exhibited inhibitory effects not only on proliferation and efflux pump of cultured cancer cells but also pathogenic biofilm formation. Phytopolyphenols (P) and MTX potentiated these inhibitory effects of Cl. In addition, novel regimens containing Cl, memantine (Mem) or thioridazine (TRZ) further enhanced not only efficacy and selectivity of anticancer effects but also inhibition on efflux pump and pathogenic biofilm formation of four chemotherapeutic agents (MTX, cisplatin, 5-fluorouracil and doxorubicin) respectively. Conclusion: In this study, novel regimens of phytopolyphenols (P), targeting drugs (T; Cl, Mem or TRZ) and metal ions (M; ZnSO4) so called PTM regimens exerted not only by themselves but also markedly potentiated efficacy and selectivity of anticancer effects of four chemotherapeutic agents. Because of their potent inhibitions on efflux pump and pathogenic biofilm formation, these combinatorial novel regimens were expected to be able to overcome the problems of multidrug resistant cancers and merit for further clinical studies.

MiR-424/TGIF2-Mediated Pro-Fibrogenic Responses in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) has been recognized as a potentially malignant disorder and is characterized by inflammation and the deposition of collagen. Among various regulators of fibrogenesis, microRNAs (miR) have received great attention but the detailed mechanisms underlying the miR-mediated modulations remain largely unknown. Here, we showed that miR-424 was aberrantly overexpressed in OSF tissues, and then we assessed its functional role in the maintenance of myofibroblast characteristics. Our results demonstrated that the suppression of miR-424 markedly reduced various myofibroblast activities (such as collagen contractility and migration ability) and downregulated the expression of fibrosis markers. Moreover, we showed that miR-424 exerted this pro-fibrosis property via direct binding to TGIF2, an endogenous repressor of the TGF-ß signaling. In addition, our findings indicated that overexpression of miR-424 activated the TGF-ß/Smad pathway, leading to enhanced myofibroblast activities. Altogether, our data revealed how miR-424 contributed to myofibroblast transdifferentiation, and targeting the miR-424/TGIF2 axis may be a viable direction for achieving satisfactory results from OSF treatment.

Depletion of miR-155 hinders the myofibroblast activities and reactive oxygen species generation in oral submucous fibrosis.

BACKGROUND/PURPOSE: Emerging evidence suggests the significance of microRNA-155 (miR-155) in fibrogenesis and oxidative stress accumulation, but its function in oral submucous fibrosis (OSF) has not been investigated. In this study, we assessed the expression of miR-155 and its effects on the maintenance of myofibroblast activation. METHODS: qRT-PCR was conducted to assess the expression of miR-155 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF samples. Collagen gel contraction, migration, and invasion capabilities were examined in fBMFs. DCFDA ROS assay was used to examine ROS generation. A luciferase reporter assay was carried out to verify the relationship between miR-155 and its potential target RPTOR. RESULTS: We showed that the expression of miR-155 was aberrantly upregulated in OSF specimens and fBMFs. Inhibition of miR-155 ameliorated various myofibroblast activities, including collagen gel contraction, migration, and invasion abilities as well as ROS production. Results from the luciferase reporter assay demonstrated that miR-155 directly interacted with its target RPTOR. CONCLUSION: Taken together, these findings indicate that miR-155 is implicated in the pathogenesis of oxidative stress-associated OSF, possibly through the regulation of RPTOR.

Silencing periostin inhibits myofibroblast transdifferentiation of fibrotic buccal mucosal fibroblasts.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) a well-recognized oral premalignant disorder. Several studies have demonstrated that periostin, a matricellular protein, is involved in the development and pathogenesis of fibrosis diseases. Nevertheless, the contribution of periostin in OSF remains to be uncovered. The purpose of the study was to illustrate the functional role of periostin involved in OSF pathogenesis. METHODS: RNA-sequencing was employed to screen for differentially expressed genes in normal and OSF tissues. Validation of the upregulation of periostin in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs) was conducted by qRT-PCR. The correlation of the gene expression of periostin and various fibrosis markers was analyzed. In addition, the functional role of periostin in myofibroblast features was tested using collagen gel contraction and transwell migration assays. RESULTS: We observed overexpression of periostin in OSF specimens using RNA-sequencing and confirmed its upregulation in OSF tissues and patient-derived fBMFs. Besides, there was a positive relationship between the expression of periostin and several fibrosis-associated markers, including ACTA2 (α-smooth muscle actin; α-SMA), COL1A1 (type 1 collagen α1 chain), TGFB1 (TGF-ß1), and FN1 (fibronectin). Furthermore, we examined the effect of silencing periostin on the maintenance of myofibroblast characteristics and showed that knockdown of periostin suppressed the expression of α-SMA. Also, inhibition of periostin markedly downregulated the myofibroblast activities (collagen gel contraction and migration capacities). CONCLUSION: Our results indicate the aberrant expression of periostin in OSF tissues and myofibroblasts. Moreover, the expression of periostin is positively associated with fibrosis markers, and repression of periostin may be a promising direction to alleviate the progression of OSF.

miR-1246 as a therapeutic target in oral submucosa fibrosis pathogenesis.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is a precancerous condition of oral cancer with a complex etiology. Our previous work has demonstrated that non-coding RNA miR-1246 contributes to the cancer stemness of oral cancer. In the current study, we sought to investigate the effect of the inhibition of miR-1246 on the oral fibrogenesis. METHODS: The expression levels of miR-1246 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined by qRT-PCR. Collagen gel contraction and migration assays were conducted to evaluate the myofibroblast activities. The relationship between miR-1246 and type I collagen was assessed and the protein expression of type I collagen was determined by Western blot. RESULTS: MiR-1246 expression was upregulated in both OSF specimen and fBMFs compared to the normal counterparts. Inhibition of miR-1246 successfully suppressed the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, the expression of miR-1246 was positively correlated with type I collagen and the expression of type I collagen was abrogated by repression of miR-1246. CONCLUSION: MiR-1246 is not only critical to the maintenance of oral stemness but also important to the activation of myofibroblasts. Our results showed that miR-1246 is positively associated with the type I collagen, which may be a downstream effector of miR-1246 and responsible for the fibrosis effect on fBMFs.

Association between Proton Pump Inhibitor Use and CNS Infection Risk: A Retrospective Cohort Study.

This study investigated the incidence of central nervous system (CNS) infection following the use of proton pump inhibitors (PPIs). A retrospective cohort study was conducted in Taiwan by using data from the National Health Insurance Research Database. We identified and enrolled 16,241 patients with CNS infection who used PPIs (PPI users). The patients were individually propensity score matched (1:1) according to age, sex, hypertension, hyperlipidemia, Charlson comorbidity index (CCI), H2 blocker, non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroid, and immunosuppressant use with 16,241 controls (PPI nonusers). A Cox proportional hazards model was used to estimate adjusted hazard ratio (aHR) for CNS infection in the PPI users and nonusers. After adjustment for other confounding factors, the incidence of CNS infection in the PPI users was 2.23-fold higher than that in the PPI nonusers (95% CI = 1.27⁻3.94). In addition, the PPI users exhibited a higher risk of CNS infection than the nonusers in the hypertension and CCI = 1 groups (aHR = 3.80, 95% CI = 1.40⁻10.32; aHR = 2.47, 95% CI = 1.07⁻5.70 in the PPI users and nonusers, respectively). In conclusions, according to these results, we concluded that the incidence of CNS infection was higher in the PPI users than in the nonusers.

miR-1246 Targets CCNG2 to Enhance Cancer Stemness and Chemoresistance in Oral Carcinomas.

MiRNAs have been recognized as crucial components in carcinogenesis, but whether miR-1246 affects the cancer stemness and drug resistance in oral squamous cell carcinoma (OSCC) has not been fully understood and its downstream targets still need to be unraveled. In the present work, we employed miRNAs RT-PCR analysis to evaluate the expression of miR-1246 in tumor tissues and oral cancer stem cells (OCSC). Stemness phenotypes, including self-renewal, migration, invasion, colony formation capacities, and in vivo oncogenicity of oral cancer cells following transfected with miR-1246 inhibitors or mimics were examined. Our results suggested that the expression level of miR-1246 was significantly upregulated in the tumor tissues and OCSC. Kaplan-Meier survival analysis of OSCC patients with high levels of miR-1246 had the worst survival rate compared to their low-expression counterparts. Inhibition of miR-1246 in OCSC significantly reduced the stemness hallmarks, while overexpression of miR-1246 enhanced these characteristics. Moreover, we showed that downregulation of miR-1246 decreased chemoresistance. In addition, we verified that miR-1246-inhibited CCNG2 contributed to the cancer stemness of OSCC. These results demonstrated the significance of miR-1246 in the regulation of OSCC stemness. Targeting miR-1246-CCNG2 axis may be beneficial to suppress cancer relapse and metastasis in OSCC patients.

GMI ablates cancer stemness and cisplatin resistance in oral carcinomas stem cells through IL-6/Stat3 signaling inhibition.

Cancer stem cells (CSCs) have been identified to exert tumor-initiating ability, resulting in the recurrence, metastasis and chemoresistance of oral squamous cell carcinomas. In the present study, we showed that GMI, an immunomodulatory protein from Ganoderma microsporum, induc ed a cytotoxic effect in oral carcinomas stem cells (OCSCs). Treatment of GMI dose-dependently inhibited the expression of CSC markers, including ALDH1 activity and CD44 positivity. Moreover, GMI suppressed the self-renewal property, colony formation, migration, and invasion abilities as well as potentiated chemo-sensitivity in OCSCs. Our results suggested that the tumor suppressive effect of GMI was mediated through inhibition of IL-6/Stat3 signaling pathway. Furthermore, tumor growth was reduced in mice bearing xenograft tumors after oral administration of GMI. Taken together, we demonstrated the anti-CSC effect of GMI in oral cancer and GMI may serve as a natural cisplatin adjuvant to prevent cancer recurrence.

DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells.

Tumor-initiating cells (TICs) are defined as a specialized subset of cells with tumor-initiating capacity that can initiate tumor growth, tumor relapse and metastasis. In the present study, osteosarcoma TICs (OS-TICs) were isolated and enriched from the osteosarcoma U2OS and MG-63 cell lines using sphere formation assays and serum-depleted media. These enriched OS-TICs showed the expression of several typical cancer stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, cluster of differentiation (CD)117, Nestin and CD133, and the expression of ATP binding cassette subfamily G member 2, multidrug resistance protein 1 (MDR1) and dihydrofolate reductase (DHFR). Notably, in vitro and in vivo tumorigenic properties were enhanced in these OS-TICs. Additionally, methotrexate and doxorubicin are the most widely used anticancer agents against osteosarcoma, and the observed enhanced chemoresistance of OS-TICs to these two agents could be associated with the upregulation of DHFR and MDR1. These findings suggest that the upregulation of DHFR and MDR1 is associated with the development of chemoresistance of OS-TICs.

Downregulation of miR-1 enhances tumorigenicity and invasiveness in oral squamous cell carcinomas.

BACKGROUND/PURPOSE: Cumulative evidence suggest that microRNAs (miRNAs) function as biosignatures of oral squamous cell carcinomas (OSCC). However, the functional roles of miR-1 as well as its downstream targets in the regulation of tumorigenicity in OSCC remain unclear. METHODS: miRNAs RT-PCR analysis was performed to identify miR-1 as a putative candidate on mediating invasiveness of OSCC cells. Consequently, we elucidated the tumorigenicity of OSCC cells with miR-1 downregulation or overexpression, respectively. Finally, miR-1 on OSCC tumor tissues was examined. RESULTS: miR-1 levels were significantly downregulated in the malignant OSCC cells. Overexpression of miR-1 significantly reduced migration/invasiveness of OSCC cells. In addition, overexpression of miR-1 decreased cancer stem cells properties. Conversely, downregulation of miR-1 promotes migration and invasiveness in OSCC cells. We have shown that miR-1 is able to target Slug, suppressing their expression. Clinically, lower miR-1 expression was found in patients with advanced nodal metastasis OSCC. CONCLUSION: miR-1 as novel biosignatures in OSCC lymph node metastatic patients, supporting the development of novel strategies for OSCC treatment.

Acquisition cancer stemness, mesenchymal transdifferentiation, and chemoresistance properties by chronic exposure of oral epithelial cells to arecoline.

Oral squamous cell carcinoma (OSCC), one of the most deadliest malignancies in the world, is caused primarily by areca nut chewing in Southeast Asia. The mechanisms by which areca nut participates in OSCC tumorigenesis are not well understood. In this study, we investigated the effects of low dose long-term arecoline (10 µg/mL, 90-days), a major areca nut alkaloid, on enhancement cancer stemness of human oral epithelial (OE) cells. OE cells with chronic arecoline exposure resulted in increased ALDH1 population, CD44 positivity, stemness-related transcription factors (Oct4, Nanog, and Sox2), epithelial-mesenchymal transdifferentiation (EMT) traits, chemoresistance, migration/invasiveness/anchorage independent growth and in vivo tumor growth as compared to their untreated controls. Mechanistically, ectopic miR-145 over-expression in chronic arecoline-exposed OE (AOE) cells inhibited the cancer stemness and xenografic. In AOE cells, luciferase reporter assays further revealed that miR-145 directly targets the 3' UTR regions of Oct4 and Sox2 and overexpression of Sox2/Oct4 effectively reversed miR-145-regulated cancer stemness-associated phenomenas. Additionally, clinical results further revealed that Sox2 and Oct4 expression was inversely correlated with miR-145 in the tissues of areca quid chewing-associated OSCC patients. This study hence attempts to provide novel insight into areca nut-induced oral carcinogenesis and new intervention for the treatment of OSCC patients, especially in areca nut users.

Corrigendum to "Caffeic Acid Phenethyl Ester Inhibits Oral Cancer Cell Metastasis by Regulating Matrix Metalloproteinase-2 and the Mitogen-Activated Protein Kinase Pathway".

[This corrects the article DOI: 10.1155/2012/732578.].

Suppression of miR-204 enables oral squamous cell carcinomas to promote cancer stemness, EMT traits, and lymph node metastasis.

The feature of oral squamous cell carcinomas (OSCC) is commonly metastasizing to locoreginal lymph nodes, and the involvement of lymph nodes metastasis represents the one of important prognostic factors of poor clinical outcome. MicroRNAs (miRNAs) have been shown to be key players of cancer-related hallmarks including cancer stemness, EMT (epithelial-mesenchymal transition), and metastaisis. Herein we showed that OSCC-derived ALDH1+ cancer stem cells (OSCC-CSCs) express lower level of miR-204, and miR-204 over-expression suppresses cancer stemness and in vivo tumor-growth of OSCC-CSCs. miR-204 binds on their 3'UTR-regions of Slug and Sox4 and suppressing their expression in OSCC-CSCs. On the contrary, down-regulation of miR-204 significantly increased cancer stemness and the lymph nodes incidence of orthotopic animal models. Furthermore, co-knockdown with sh-Slug and sh-Sox4 synergistically rescued miR-204-supressing cancer stemness and EMT properties. Clinical results further revealed that a miR-204lowSlughighSox4high signature predicted the worse survival prognosis of OSCC patients by Kaplan-Meier survival analyses. Up-regulated miR-204-targeting Slug and Sox4 by epigallocatechin-3-gallate (EGCG) treatment significantly inhibited the proliferation rate, self-renewal capacity, and the percentage of ALDH1+ and CD44+ cells in OSCC-CSCs Oral-feeding of EGCG effectively alleviated tumor-progression in OSCC-CSCs-xenotransplanted immunocompromised mice through miR-204 activation. In conclusion, miR-204-mediated suppression of cancer stemness and EMT properties could be partially augmented by the anti-CSCs effect of EGCG.

Targeting CD133 in the enhancement of chemosensitivity in oral squamous cell carcinoma-derived side population cancer stem cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. Previously, we enriched a subpopulation of OSCC-derived cancer stem cells (OSCC-CSCs), and identified CD133 as an OSCC-CSC marker. METHOD: We determined the function of CD133 on chemosensitivity of oral cancer CSCs by silencing CD133. RESULTS: Initially, we observed that the expression profile of CD133 in OSCC-side population (OSCC-SPs) cells, which exerted properties of CSCs, was significantly upregulated than that of major population (MPs) cells of OSCCs. The cell viability experiments showed that SPs were more chemoresistant compared with major populations. Importantly, targeting CD133 ameliorated the drug resistance of OSCC-SPs to cisplatin treatment. Targeting CD133 and cisplatin co-treatment led to the maximal inhibition on tumor initiating properties in OSCC-SPs. CONCLUSION: Side population cells with CSCs properties existed in OSCCs, and silencing CD133 exhibited a prominent therapeutic effect in enhancing the sensitivity of chemotherapy in OSCC through elimination of CSCs. © 2015 Wiley Periodicals, Inc. Head Neck 38: E231-E238, 2016.

Periodontal status in Taiwanese pregnant women.

BACKGROUND/PURPOSE: Few studies have investigated the periodontal status of Taiwanese pregnant women. This study aimed to investigate the periodontal status of pregnant women and to examine its relation to oral hygiene. MATERIAL AND METHODS: This study randomly recruited 477 pregnant women. Among them, 203 women were in their first trimester. Forty-six women completed the study to the end of their third trimester. We also recruited 160 nonpregnant women as the control group. Clinical periodontal parameters were recorded and included probing pocket depth [PPD (mm)], clinical attachment level [CAL (mm)], gingival index simplified [GI-s (%)], and plaque index [PI (%)]. RESULTS: The GI-s of the pregnant group (PG) was higher than that of the control group [CG; (i.e., nonpregnant)], but only the third trimester was statistically significantly different (P < 0.001).The full mouth dental PI was higher in the PG than in the CG (P < 0.001), particularly in the interproximal areas. The mean PPD was greater in the PG than in the CG (P < 0.001) in all tooth areas. The mean CAL was higher in the PG than in the CG (P < 0.001), but no difference existed between the different trimesters. The CG had a higher percentage of sites with a shallow PPD, compared to the PG (P < 0.001); the PG had a higher percentage of sites with a PPD of 4-6 mm, compared to the CG (P < 0.001). Only the PI of the full mouth and lingual tooth surfaces in the third trimester were better than in the first trimester throughout the pregnancy. CONCLUSION: Gingival inflammation in pregnant women is positively correlated with the increased deposition of a dental plaque biofilm.

Knockdown of S100A4 impairs arecoline-induced invasiveness of oral squamous cell carcinomas.

OBJECTIVES: Metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. The physiological function of S100A4 in the pathogenesis of areca quid chewing-associated OSCC has not been uncovered. METHOD: OSCC tissues from areca quid chewers were analyzed by immunohistochemistry for S100A4 expression. The functions of S100A4 in invasiveness of arecoline-treated oral epithelial (OE) cells were determined by loss function approaches. RESULTS: Expression of S100A4 was positively correlated with clinical grading and lymph node metastasis of OSCC. Upregulated S100A4 is correlated with poor survival outcome of OSCC patients. Arecoline led to dose-dependent elevation of S100A4 expression in oral epithelial (OE) cells. Down-regulation of S100A4 significantly reversed arecoline-induced oncogenecity in OE cells. The additions of pharmacological agents LY294002, SP600125, and CAY10585 were found to inhibit arecoline-induced S100A4 expression in OE cells. CONCLUSION: Arecoline-induced S100A4 expression was down-regulated by LY294002, SP600125, or CAY10585 treatment. Targeting S100A4 might offer a new strategy for the treatment of OSCC patients with metastasis.

Elevated Lin28B expression is correlated with lymph node metastasis in oral squamous cell carcinomas.

BACKGROUND: Lymph node (LN) metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. Searching the detailed molecular mechanisms involved LN metastasis in OSCC is still an open question. METHODS: Paired tissue samples from tumor (T) and adjacent non-cancerous matched tissues (NCMT) parts, as well as LN metastatic lesions in patient with OSCC tissues were subjected to quantitative real-time PCR analysis for the expression levels of Lin28B. Arecoline, a major areca nut alkaloid, was to explore whether expression of Lin28B could be changed dose dependent in oral epithelial cells. Control and Lin28B-knockdown arecoline-stimulated oral epithelial cells were subjected to migration/invasion/anchorage-independent growth assay. RESULTS: Compared with NCMT samples from the same OSCC patient, the expression of Lin28B was increased in all of the tumor samples. A similar upregulation of Lin28B was also observed in LN metastatic when compared with local tumors. Arecoline treatment dose dependently induced Lin28B expression in SG and FaDu cells. Lentiviral-mediated silencing Lin28B expression significantly attenuated arecoline-induced oncogenicity including proliferation, migration, invasiveness, and anchorage-independent growth in SG and FaDu cells. CONCLUSIONS: Lin28B may be a useful biomarker and novel molecular target for LN metastasis OSCC patients' treatment.

Resveratrol suppresses TPA-induced matrix metalloproteinase-9 expression through the inhibition of MAPK pathways in oral cancer cells.

BACKGROUND: Naturally occurring agents, such as resveratrol, have been determined to benefit health. Numerous studies have demonstrated that resveratrol has antioxidative, cardioprotective, and neuroprotective properties. However, the effect of resveratrol exerts on the metastasis of oral cancer cells remains unclear. In this study, we investigated the effect the anti-invasive activity of resveratrol on a human oral cancer cell line (SCC-9) in vitro and the underlying mechanisms. METHODS: Cell viability was examined by MTT assay, whereas cell motility was measured by migration and wound-healing assays. Zymography, reverse-transcriptase polymerase chain reaction (PCR), and promoter assays confirmed the inhibitory effects of resveratrol on matrix metalloproteinase-9 (MMP-9) expression in oral cancer cells. RESULTS: We established that various concentrations (0-100 µM) of resveratrol inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration capacities of SCC-9 cells and caused no cytotoxic effects. Zymography and Western blot analyses suggested that resveratrol inhibited TPA-induced MMP-9 gelatinolytic activity and protein expression. In addition, the results indicated that resveratrol inhibited the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 and extracellular-signal-regulated kinase (ERK)1/2 involved in downregulating protein expression and the transcription of MMP-9. CONCLUSION: In summary, resveratrol inhibited MMP-9 expression and oral cancer cell metastasis by downregulating JNK1/2 and ERK1/2 signals pathways and, thus, exerts beneficial effects in chemoprevention.