Zheng-Jun Shang

Ephrin-A1 is up-regulated by hypoxia in cancer cells and promotes angiogenesis of HUVECs through a coordinated cross-talk with eNOS.

Hypoxia, ephrin-A1 and endothelial nitric oxide synthase (eNOS) have been proved to play critical roles in tumor angiogenesis. However, how ephrin-A1 is regulated by hypoxia and whether ephrin-A1 cooperates with eNOS in modulation of angiogenesis remain to be addressed in details. Here we demonstrated that both ephrin-A1 in squamous cell carcinoma cells (SCC-9) and especially soluble ephrin-A1 in the supernatants were up-regulated under hypoxic condition. An increased nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) was observed in ephrin-A1-induced angiogenesis which was reversed after co-culture with eNOS specific inhibitor, N-nitro-L-arginine methyl ester hydrochloride (L-NAME). Western blot analysis confirmed that both phosphorylation of AktSer473 and eNOSSer1177 were up-regulated in ephrin-A1-stimulated HUVECs, with the total eNOS expression unchanged. The specific inhibitor of phosphatidylinositol 3-kinase (PI3K), LY294002, significantly down-regulated ephrin-A1-induced expression of phosphorylated AktSer473 as well as phosphorylation of eNOSSer1177. These results revealed a possible novel mechanism whereby ephrin-A1 is regulated in tumor microenvironment and promotes angiogenesis through a coordinated cross-talk with PI3K/Akt-dependent eNOS activation which may relate to normal vascular development and tumor neovascularization.

EphA2/EphrinA1 mRNA Expression and Protein Production in Adenoid Cystic Carcinoma of Salivary Gland

PURPOSE EphA2/ephrinA1 is believed to play a role in tumor growth and metastasis. The purpose of the present study was to determine the presence of EphA2/ephrinA1 in mRNA and protein adenoid cystic carcinoma. MATERIALS AND METHODS mRNA and protein expression and protein product of EphA2 and ephrinA1 in adenoid cystic carcinoma was investigated using real-time reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry. The tyrosine-phosphorylated state of EphA2 in adenoid cystic carcinoma cells was also investigated. RESULTS Greater expression of EphA2 and ephrinA1 proteins and mRNA was detected in adenoid cystic carcinoma tissues. EphA2/ephrinA1 staining activities in adenoid cystic carcinoma were more significant than those in normal gland tissue (P < .01). EphA2/ephrinA1 expression correlated significantly to the microvessel density (P < .01). EphA2/ephrinA1 expression and microvessel density correlated with the clinical TNM stage, perineural invasion, and vascular invasion (P < .05). In 3 histologic types of adenoid cystic carcinoma, the expression of EphA2/ephrinA1 and microvessel density was significantly greater in the solid type than in the cribriform and tubular types (P < .01). We also noted that EphA2 was present in a nontyrosine-phosphorylated state. CONCLUSIONS The present study showed a high expression of EphA2/ephrinA1 in adenoid cystic carcinoma. EphA2/ephrinA1 can serve as a novel therapy target for adenoid cystic carcinoma.

Tumor necrosis factor-α enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway.

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-α (TNF-α) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-α-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-α could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis.

Correlation between CCR7 expression and lymph node metastatic potential of human tongue carcinoma

Metastasis is an important cause of cancer-related mortality. In this study, we investigated the role of CCR7 in the lymph node metastasis of tongue carcinoma. Immunohistochemistry and Western blot revealed the expression of CCR7 in tongue SCC tissues and cell lines. In addition, we examined the expression of CCL21, a ligand of CCR7, in normal and diseased lymph nodes using immunohistochemistry and/or real-time PCR. The CCR7 expression was significantly correlated with cervical lymph node metastasis, tumor staging, and histological grade (P = 0.015, 0.040, and 0.015, respectively). The multivariate analysis showed that regional lymph node metastasis, the expression of CCR7, and LVD were the independent poor prognostic factors. Knockdown of CCR7 gene resulted in a significant inhibition of migration and invasion of SCC4 cells in vitro without affecting the proliferation and apoptosis of tumor cells. Also, CCR7 knockdown obviously inhibited cervical lymph node metastasis in an animal tumor model. Our study indicated that CCR7 may play an important role in progression of tongue SCC and could be a promising target for tongue SCC therapy.

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.

Alterations of high endothelial venules in primary and metastatic tumors are correlated with lymph node metastasis of oral and pharyngeal carcinoma.

High endothelial venules (HEVs) are special blood vessels in the paracortical region of lymph nodes (LNs) and govern lymphocyte recruitment. LN metastasis has similarity to circulating lymphocytes homing to LNs, but the role of HEVs in the progression of oral and pharyngeal squamous cell carcinoma (OPSCC) is unclear. In this study, we found that HEVs experienced a series of morphological and functional changes during OPSCC progression and were correlated with LN metastasis. In 9 cases of 73 metastatic LNs, tumor emboli were located adjacent to HEVs or just out of the vessels but not lymphatic channels. Gap junctions of tumor cells close to HEVs decreased or disappeared, and gaps were left at contact points where tumor cells attached to the HEVs. Moreover, the proliferation rate of endothelial cells of HEVs was the highest in metastatic LNs. Finally, L-selectin was detected in both primary and metastatic tumors, and it facilitated tumor cells adhering to LNs. In conclusion, our findings suggest that remodeled HEVs are correlated with LN metastasis of OPSCC and play important role in this process by preparing premetastatic soil for cancer cell metastasis.

Up-regulation of syncytin-1 contributes to TNF-α-enhanced fusion between OSCC and HUVECs partly via Wnt/β-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/β-catenin signal pathway in SCC-9. The activation of Wnt/β-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/β-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/β-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.

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.

Clinical experience with 80 microvascular couplers in 64 free osteomyocutaneous flap transfers for mandibular reconstruction.

Microvascular couplers have been introduced as an alternative method for anastomosis in mandibular reconstruction. This study included 64 patients who had undergone free flap reconstruction for mandibular defects and had been scheduled for follow-up at 1, 3, 6, and 12 months. After completion of the tumour resection and harvesting of the osteomyocutaneous flap, appropriate preparation of both ends of the vessels was performed for microsurgery. Single-vein anastomoses were performed in 35 patients and double-vein anastomoses in 29 patients. Except for 75 couplers used for venous anastomosis only, both arterial and venous anastomoses were performed using the coupler in seven flaps. No flap failures occurred in these cases, resulting in an overall flap success rate of 100%. As expected, anastomoses were completed successfully using the coupler in 78 out of 80 attempted cases (97.5%). Additional large and randomized studies are needed to compare the outcomes of coupler anastomoses to those of traditional sutured anastomoses, and to define to what extent this would present cost-savings per procedure.

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.