Autophagy Induced by Areca Nut Extract Contributes to Decreasing Cisplatin Toxicity in Oral Squamous Cell Carcinoma Cells: Roles of Reactive Oxygen Species/AMPK Signaling.

Chewing areca nut is closely associated with oral squamous cell carcinoma (OSCC). The current study aimed to investigate potential associations between areca nut extract (ANE) and cisplatin toxicity in OSCC cells. OSCC cells (Cal-27 and Scc-9) viability and apoptosis were analyzed after treatment with ANE and/or cisplatin. The expressions of proteins associated with autophagy and the AMP-activated protein kinase (AMPK) signaling network were evaluated. We revealed that advanced OSCC patients with areca nut chewing habits presented higher LC3 expression and poorer prognosis. Reactive oxygen species (ROS)-mediated autophagy was induced after pro-longed treatment of ANE (six days, 3 µg). Cisplatin toxicity (IC50, 48 h) was decreased in OSCC cells after ANE treatment (six days, 3 µg). Cisplatin toxicity could be enhanced by reversed autophagy by pretreatment of 3-methyladenine (3-MA), N-acetyl-l-cysteine (NAC), or Compound C. Cleaved-Poly-(ADP-ribose) polymerase (cl-PARP) and cleaved-caspase 3 (cl-caspase 3) were downregulated in ANE-treated OSCC cells in the presence of cisplatin, which was also reversed by NAC and Compound C. Collectively, ANE could decrease cisplatin toxicity of OSCC by inducing autophagy, which involves the ROS and AMPK/mTOR signaling pathway.

Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression.

Hypoxia Enhances Fusion of Oral Squamous Carcinoma Cells and Epithelial Cells Partly via the Epithelial-Mesenchymal Transition of Epithelial Cells.

Increasing evidence and indications showed that cell fusion is crucial in tumor development and metastasis, and hypoxia, a closely linked factor to tumor microenvironment, which can lead to EMT, induces angiogenesis and metastasis in tumor growth. However, the relationship between hypoxia and fusion has not been reported yet. EMT will change some proteins in the epithelial cell surface and the changes of proteins in cell surface may increase cell fusion. This study found that hypoxia promotes the spontaneous cell fusion between Oral Squamous Carcinoma Cells (OSCCs) and Human Immortalized Oral Epithelial Cells (HIOECs). At the same time, Hypoxia can lead to EMT, and hypoxia-pretreated HIOECs increased fusion rate with OSCC, while the fusion rate was significantly reduced by DAPT, a kind of EMT blocker. Therefore, epithelial cells can increase spontaneously cell fusion with OSCC by EMT. Our study may provide a new insight to link among tumor microenvironment, cell fusion, and cancer.

The Toll-like receptor 4 antagonist TAK-242 protects against chronic pancreatitis in rats.

Chronic pancreatitis is a progressive disease characterized by irreversible morphological changes to the pancreas, typically causing pain and permanent loss of function. It is a poorly understood disease with the pathogenesis remaining unclear. The authors' previous data demonstrated that the inhibition of Toll­like receptor 4 (TLR4) using TLR4 antagonist kinase (TAK)­242 attenuates taurocholate­induced oxidative stress via the regulation of mitochondrial function in the pancreatic acinar cells of mice. In the present study, the effect of TAK­242 on trinitrobenzene sulfonic acid (TNBS)­induced chronic pancreatitis was investigated in rats. The results revealed that TAK­242 attenuated the severity of chronic pancreatic injury, and regulated extracellular matrix secretion and cellular immunity. In addition, TAK­242 treatment significantly decreased cell apoptosis, as evidenced by the reduction in Terminal deoxynucleotidyl transferase dUTP nick end labeling­positive cells in pancreas tissue sections, and also promoted cell proliferation in TNBS­treated animals. Furthermore, the results of the calibrated von Frey filament assay demonstrated that TAK­242 could prevent the pancreatitis­induced referred abdominal hypersensitivity. In summary, TAK­242 exhibits protective effects against TNBS­induced chronic pancreatitis and may be a potential therapeutic strategy for the treatment of patients with chronic pancreatitis.

Up-regulation of syncytin-1 contributes to TNF-α-enhanced fusion between OSCC and HUVECs partly via Wnt/ß-catenin-dependent pathway.

Accumulating evidence implies that cell fusion is one of the driving forces of cancer invasion and metastasis. However, considerably less is still known about the triggering factors and underlying mechanisms associated with cancer-host cell fusion, particularly in inflammatory tumor microenvironment. In this study, we confirmed that inflammatory factor TNF-α could enhance fusion between squamous cell carcinoma cells 9 (SCC-9) and human umbilical vein endothelial cells (HUVEC). Further study revealed that TNF-α could promote up-regulation of syncytin-1 in SCC-9 and its receptor neutral amino acid transporter type 2 (ASCT-2) in HUVEC. Syncytin-1 acted as an important downstream effector in TNF-α-enhanced cancer-endothelial cell fusion. TNF-α treatment also led to the activation of Wnt/ß-catenin signal pathway in SCC-9. The activation of Wnt/ß-catenin signal pathway was closely associated with the up-regulation of syncytin-1 in SCC-9 and increased fusion between SCC-9 and HUVEC while blocking of Wnt/ß-catenin signal pathway resulted in the corresponding down-regulation of syncytin-1 accompanied by sharp decrease of cancer-endothelial cell fusion. Taking together, our results suggest that Wnt/ß-catenin signal pathway activation-dependent up-regulation of syncytin-1 contributes to the pro-inflammatory factor TNF-α-enhanced fusion between oral squamous cell carcinoma cells and endothelial cells.

EphA2 silencing promotes growth, migration, and metastasis in salivary adenoid cystic carcinoma: in vitro and in vivo study.

EphA2 is associated with tumor growth and distant metastasis in numerous human tumors. Considering the controversial effects of EphA2 in different tumors and the lack of reports in salivary adenoid cystic carcinoma (SACC), we evaluated the effects of EphA2 inhibition by short hairpin RNA on SACC through in vivo and in vitro researches for the first time. Real-time reverse transcriptase-PCR and western blot analysis were conducted to verify the interference effect on SACC cells. Using Cell Counting Kit-8, wound healing, Transwell and Matrigel adhesion assays, we confirm that inhibition of EphA2 promotes the migration, invasion and adhesion ability of SACC cells. In vivo research, we prove that silencing of EphA2 significantly accelerates tumor growth and lung metastasis ability by establishing xenograft models in mice, including subcutaneous inoculation and tail vein injection. In addition, immunostaining of EphA2, E-cadherin and Slug from 40 specimens and in vitro simulation of perineural invasion (PNI) assay imply that suppression of EphA2 partially contribute to epithelial-mesenchymal transition and enhancement of PNI in SACC. In conclusion, all the data suggest that EphA2 may act as a tumor suppressor in SACC progression.

Membrane microvesicles as mediators for melanoma-fibroblasts communication: roles of the VCAM-1/VLA-4 axis and the ERK1/2 signal pathway.

Recent studies suggest that microvesicles (MVs) within the tumor microenvironment are emerging as potent mediators for cell-cell communication. In this study, we investigated the MV-mediated transformation of normal fibroblasts to tumor-associated fibroblasts, focusing on the functional regulation of vascular cell adhesion molecule-1 (VCAM-1) expression. After incubation with melanoma-derived MVs, the fibroblasts (NIH/3T3 cells) presented an obvious enhancement of VCAM-1 expression in an ERK1/2-activation-dependent manner, and this enhancement was further increased when the MVs were from highly metastatic melanoma cells. The adhesion analysis showed that the VCAM-1/VLA-4 axis is involved in the preferential attachment of highly metastatic melanoma cells and BMSCs to MV-educated fibroblasts. Hypoxia promoted melanoma-fibroblast interaction by directly upregulating VLA-4 expression in highly metastatic melanoma cells and indirectly provoking VCAM-1 expression in fibroblast cells via melanoma-released MVs. Moreover, MV-educated fibroblasts increased IL-6, fibroblast activation protein and EGF expression simultaneously. Proteomic analysis of MVs suggested that numerous signal pathways in addition to the MAPK signal pathway are regulated by melanoma MVs, which function as tumor messengers that participate in melanoma progression.

Dopamine D3 receptor-regulated NR2B subunits of N-methyl-d-aspartate receptors in the nucleus accumbens involves in morphine-induced locomotor activity.

AIMS: Dopamine and glutamate receptors are densely expressed in the nucleus accumbens (NAc). Active interactions between these receptors contribute to the development of neuropsychiatric diseases, such as drug addiction and relapse. However, the molecular mechanisms underlying these interactions remain unclear. METHODS: This study established a mouse model of intermittent morphine-induced mouse behavioral sensitization model. Western blot and electrophysiological recording methods were performed to directly identify the affective components of morphine behavioral sensitization. RESULTS: Interval morphine administration could cause significant locomotor sensitization. Hyperlocomotion and behavioral locomotor sensitization were significantly suppressed when ifenprodil (5 mg/kg), a selective NR2B subunit-containing N-methyl-d-aspartate (NMDA) receptor antagonist, or nafadotride (25 µg/kg), a dopamine D3 receptor (D3R)-preferring antagonist, was coadministered with morphine. Western blot analysis showed that morphine behavioral sensitization induced a region-specific increase in phosphorylation of NR2B (pNR2B) and total levels of NR2B (NR2B) expression in the NAc. Systemically administered nafadotride attenuated behavioral locomotor sensitization induced by morphine and significantly reversed the overexpression of pNR2B and NR2B subunit-containing NMDA receptor in the NAc. NMDA receptor-mediated excitatory postsynaptic currents in the NAc were also significantly reduced by nafadotride. CONCLUSIONS: These findings suggest that D3Rs are involved in morphine-induced behavioral locomotor sensitization in mice by regulating the NR2B subunits of NMDA receptors in the NAc.