Guiquan Zhu

Hypoxia-inducible factor-1 alpha, in association with TWIST2 and SNIP1, is a critical prognostic factor in patients with tongue squamous cell carcinoma

It has become apparent that hypoxia and hypoxia-inducible factor-1 (HIF-1) activation have the potential of modulating the activity of major epithelial-mesenchymal transition (EMT)-triggering pathways by regulating the expression and activity levels of major transcriptional repressors. The aim of our study was to elucidate the role of HIF-1α and HIF-2α, and EMT regulators TWIST2 and SMAD nuclear interacting protein-1 (SNIP1) in tongue squamous cell carcinoma (TSCC). A retrospective analysis of 89 patients with TSCC from Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University between 2002 and 2005 was performed using immunohistochemistry in paraffin-embedded and formalin-fixed tissues to analyze HIF-1α, HIF-2α, TWIST2 and SNIP1 expression. The association between HIF-1α, HIF-2α, TWIST2 and SNIP1 expression and patient survivals was investigated. Our results showed that overexpression of HIF-1α, HIF-2α, TWIST2 and SNIP1 were shown in 49.44% (44/89), 55.06% (49/89), 44.94% (40/89) and 34.83% (31/89) of TSCC, respectively. Overexpression of HIF-1α, TWIST2 and SNIP1 in TSCC was associated with a shorter disease-free survival (P=0.003, P=0.001, P=0.040, respectively), and HIF-2α had no significant association with either overall survival (P=0.195) or disease-free survival (P=0.356). Co-expression of more than two markers of HIF-1α, TWIST2 and SNIP1 was an independent prognostic indicator for both overall survival and disease-free survival by multivariate Cox proportional hazards model. It is proposed that co-expression of more than two markers from HIF-1α, TWIST2 and SNIP1 might be a significant prognostic predictor in patients with TSCC.

Expression of c-kit and Slug correlates with invasion and metastasis of salivary adenoid cystic carcinoma

The overexpression of c-kit seems to be frequent and specific in salivary adenoid cystic carcinoma (ACC), however, there is little information on correlation between c-kit expression and the invasion and metastasis. Recently, the data showed that Slug, a transcription factor of epithelial-mesenchymal transitions (EMT), is a molecular target that contributes to the biological specificity of c-kit signaling pathway. In this study, the expression of c-kit and Slug was evaluated in two ACC cell lines and 121 patients with ACC. The results of real-time RT-PCR and Western blot showed that ACC-2 and ACC-M cell lines expressed c-kit and Slug mRNA and protein. The immunohistochemical assay in patients demonstrated that positive expression of c-kit and Slug was observed in 108/121 (89.26%) and 87/121 (71.90%) of cases, respectively, and that c-kit and Slug expression was significantly associated with tumor site, TNM stage, histological pattern, perineural invasion, local regional recurrence and distant metastasis of patients with ACC (P<0.05). Furthermore, there was a significant association between the positive expression of c-kit and that of Slug (P=0.046). These findings indicated that c-kit/Slug pathway might participate in the invasion and metastasis of salivary ACC.

Hypoxia Inducible Factor 1α and Hypoxia Inducible Factor 2α Play Distinct and Functionally Overlapping Roles in Oral Squamous Cell Carcinoma

Purpose: This study aimed to investigate the functional difference between hypoxia inducible factor (HIF)-1α and HIF-2α in oral squamous cell carcinomas (OSCC). Experimental Design: We evaluated the correlations between HIF-1α and HIF-2α expression and the clinical-pathologic characteristics of 97 patients with OSCC by immunohistochemical staining. OSCC cell lines transfected with lentivirus encoding short hairpin RNA against HIF-1α/2α were used to investigate the HIF-1α/2α–dependent target genes. Xenograft tumors in nude mice were established using cells affected by lentivirus, and tumor growth, angiogenesis, proliferation, and apoptosis were measured. Results: HIF-1α expression was significantly associated with T stage (P = 0.004), lymph node involvement (P = 0.006), histologic differentiation (P = 0.013), and microvessel density (P = 0.014), whereas that of HIF-2α was associated with T stage (P = 0.011) and microvessel density (P = 0.005). Patients with positive HIF-1α nuclear staining had a significantly worse overall survival (P < 0.001) and disease-free survival (P < 0.001) than those with negative HIF-1α staining. When OSCC cells were cultured at 5% O2, only HIF-2α contributed to the expression of vascular endothelial growth factor. At 1% O2, vascular endothelial growth factor was regulated by both HIF-1α and HIF-2α, but glucose transporter 1, carbonic anhydrase 9, and urokinase-type plasminogen activator receptor were regulated by HIF-1α rather than by HIF-2α. Knocking down HIF-1α or HIF-2α individually inhibited the xenograft tumor angiogenesis and growth, and knocking them down simultaneously revealed a better inhibitory effect than knocking down either unit alone. Conclusions: HIF-1α and HIF-2α correlated with different clinical-pathologic parameters, stabilized at different oxygen levels, and regulated different genes in OSCC. However, both HIF-1α and HIF-2α showed promoting roles in tumor angiogenesis and growth, and therapeutic outcome may benefit from combined targeting of HIF-1α and HIF-2α. Clin Cancer Res; 16(19); 4732–41. ©2010 AACR.

RNAi targeting urokinase-type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo

It has been admitted that urokinase‐type plasminogen activator receptor (u‐PAR) is overexpressed in many human malignant tumors including oral squamous cell carcinoma (OSCC) and plays an important role in a variety of cancer key cellular events as a versatile signaling orchestrator. In our study, a retroviral vector expressing u‐PAR‐specific siRNA was injected into OSCC xenografts of nude mice to observe its inhibitory effects on OSCC. Our data demonstrate that siRNA targeting u‐PAR markedly suppressed tumor growth, reduced the expression of proliferation‐related gene, Ki‐67 and increased cell apoptosis, accompanying with the efficient and specific inhibition of endogenous u‐PAR expression in OSCC. More importantly, the mRNA and protein expression of MMP‐2, MMP‐9, VEGF‐C, VEGF‐D and VEGFR‐3 that are intimately involved in oral cancer invasion and metastasis, was simultaneously downregulated significantly as determined by quantitative real‐time RT‐PCR, Western blot and immunohistochemistry; and Gelatin and fibrin zymography showed that MMP‐9, MMP‐2 and u‐PA enzymatic activities were significantly reduced in u‐PAR‐specific siRNA group, compared to those in control groups. In addition, the expression of MDR‐1 gene related to drug resistance was obviously inhibited by silencing of u‐PAR. These findings suggest that RNAi targeting u‐PAR could effectively inhibit the metastasis and progression of OSCC in vivo. Thus, it may be used as a potent and specific therapy for oral cancer, especially in inhibiting and preventing cancer cell invasion and metastasis.

HIF-α/MIF and NF-κB/IL-6 Axes Contribute to the Recruitment of CD11b+Gr-1+ Myeloid Cells in Hypoxic Microenvironment of HNSCC

CD11b+Gr-1+ myeloid cells have gained much attention due to their roles in tumor immunity suppression as well as promotion of angiogenesis, invasion, and metastases. However, the mechanisms by which CD11b+Gr-1+ myeloid cells recruit to the tumor site have not been well clarified. In the present study, we showed that hypoxia could stimulate the migration of CD11b+Gr-1+ myeloid cells through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Hypoxia-inducible factor-1α (HIF-1α)- and HIF-2α-dependent MIF regulated chemotaxis, differentiation, and pro-angiogenic function of CD11b+Gr-1+ myeloid cells through binding to CD74/CXCR2, and CD74/CXCR4 complexes, and then activating p38/mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases (PI3K)/AKT signaling pathways. Knockdown (KD) of HIF-1α and HIF-2α in HNSCC cells decreased MIF level but failed to inhibit the CD11b+Gr-1+ myeloid cell migration, because HIF-1α/2α KD enhanced nuclear factor κB (NF-κB) activity that increased IL-6 secretion. Simultaneously blocking NF-κB and HIF-1α/HIF-2α had better inhibitory effect on CD11b+Gr-1+ myeloid cell recruitment in the hypoxic zone than individually silencing HIF-1α/2α or NF-κB. In conclusion, the interaction between HIF-α/MIF and NF-κB/IL-6 axes plays an important role in the hypoxia-induced accumulation of CD11b+Gr-1+ myeloid cells and tumor growth in HNSCC.

Coexpression of hypoxia-inducible factor-2α, TWIST2, and SIP1 may correlate with invasion and metastasis of salivary adenoid cystic carcinoma.

BACKGROUND Adenoid cystic carcinoma (ACC) of salivary gland is characterized by advanced local invasion and distant metastasis. Intratumoral hypoxia was reported to be associated with epithelial-mesenchymal transition (EMT) regulators. The purpose of this study was to evaluate the relationship between hypoxia-inducible factor (HIF)-2α, TWIST2, and SIP1 expression and the invasion and metastasis in ACC of salivary gland. METHOD In vitro we first detected the expression of HIF-2α, TWIST2, and SIP1 in two ACC cell lines by Western blot and real-time RT-PCR. Then, in vivo, a retrospective investigation of 121 patients with ACC from Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University between 1996 and 2005 was carried out using immunohistochemistry to analyze the association between the expression of these three factors and clinical-pathological factors of patients. RESULTS The protein and mRNA levels of HIF-2α, TWIST2, and SIP1 in the high-metastasis cell line (ACC-M) were much higher than those of the low-metastasis cell line (ACC-2). The positive expression of HIF-2α, TWIST2, and SIP1 (71.07%, 42.98%, and 38.02%, respectively) was associated with the perineural invasion, the local recurrence, and distant metastasis of patients with ACC (P < 0.05). The patients with the positive coexpression of the three factors had a lower survival rate than those with the negative expression (P < 0.05). CONCLUSION It is proposed that the elevated expression of HIF-2α, TWIST2, and SIP1 can contribute to invasion and metastasis of ACC, and there might be some correlation between the hypoxia microenvironment and EMT in ACC.

Correlation between transcription factor Snail1 expression and prognosis in adenoid cystic carcinoma of salivary gland.

OBJECTIVE Transcription factor Snail1 has a central role in induction of epithelial-mesenchymal transition (EMT). The aim of the present study was to study the association of Snail1 expression with clinicopathologic factors and prognosis in adenoid cystic carcinoma (ACC) of salivary gland. STUDY DESIGN Demographic variables and primary tumor site, dates of diagnoses, perineural invasion, local regional recurrence, and distant metastasis of 121 cases of salivary ACC were retrieved. The Snail1 expression of all these cases was examined by immunohistochemical methods. The association between Snail1 expression and clinicopathologic variables was analyzed using chi-square test, and the prognostic factors were examined by univariate and multivariate analyses. RESULTS Our data demonstrated that the positive expression of Snail1 was observed in 58.68% (71/121) of cases. Snail1 expression was significantly associated with histologic pattern, perineural invasion, local regional recurrence, and distant metastasis of patients with ACC (P < .05). And Snail1 expression, local regional recurrence, and distant metastasis were independent and significant prognostic factors in all patients. CONCLUSION Snail1 may play an important role in the invasion and metastasis of ACC, and Snail1 has the potential for being a strong prognosis indicator at presentation of ACC patients.

Maxillofacial space infection experience in West China: a retrospective study of 212 cases

OBJECTIVES Our objectives were to analyze the clinical features of maxillofacial space infection (MSI) patients admitted to the West China Hospital of Stomatology over a five-year period, and to identify potential risk factors associated with life-threatening complications. METHODS A retrospective medical chart review was performed and the sociodemographic and clinical characteristics of patients with MSI were evaluated. RESULTS A total of 212 patients were enrolled in this study, including 125 males (59.0%) and 87 females (41.0%), with an age range of 1-88 years (median 47.5 years). The most common cause of MSI was odontogenic infection (56.1%). The submandibular space was the space most commonly involved in both single space and multiple space infections (37.5% and 29.1%, respectively). One hundred and two patients (48.1%) self-medicated before admission, and the time from onset of symptoms until presentation was longer in those who self-medicated compared with those who did not (p=0.028). Fifty-seven patients (26.9%) had life-threatening complications and six died (2.8%). In multivariate analysis, age, self-medication, admission temperature, respiratory difficulty, and underlying diseases were found to be risk factors for life-threatening complications. The most common occupation of the patients was farmer (54.7%). Among the farmers, 72.4% had an odontogenic etiology; however, 91.7% of the farmers with odontogenic space infections had not undergone dental treatment before admission. CONCLUSIONS Our experience suggests that the management of MSI should be more aggressive when the above risk factors are present, in order to avoid life-threatening complications. In addition, considering the poor medical conditions in the rural areas of West China, standard dental care and services should be provided in the future to replace self-medication.

Effects of intra-articular administration of sodium hyaluronate on plasminogen activator system in temporomandibular joints with osteoarthritis

OBJECTIVE The aim of this study was to investigate the effect of intra-articular sodium hyaluronate (SH) injections on the main components of plasminogen activator (PA) system in the synovial fluid of temporomandibular joint (TMJ) osteoarthritis (OA). STUDY DESIGN Forty patients diagnosed with TMJ OA and 20 healthy control subjects were included in this study. Synovial fluid was collected in the OA group and the healthy group at baseline. The OA patients were randomly divided into 2 groups (20 patients for each group): One group received 5 injections of SH, and the other received 5 injections of physiologic saline solution in the upper joint space at weekly intervals. Synovial fluid was collected before and after treatment. Urokinase-type PA (uPA), soluble uPA receptor (suPAR) and PA inhibitor 1 (PAI-1) levels in synovial fluid were quantified by enzyme-linked immunosorbent assay. RESULTS The OA patients had significantly higher uPA activity and levels of uPA (median 80.01 ng/L), suPAR (median 7.54 ng/L), and PAI-1 (median 54.9 ng/mL) than the healthy control subjects (median 20.47 ng/L uPA, 2.34 ng/L suPAR, and 19.9 ng/mL PAI-1; (P < .05). The uPA activity and levels of uPA, suPAR, and PAI-1 were significantly decreased after SH injections in TMJs of OA patients (P < .05), and there was no difference after saline injection. Visual analog pain score reduction correlated with changes in uPA and uPAR levels as well as uPA activity. CONCLUSION The effects of SH on PA system provide new insight into a possible underlying mechanism by which SH alleviates pain of patients with TMJ OA.

Expression and importance of zinc-finger transcription factor Slug in adenoid cystic carcinoma of salivary gland.

BACKGROUND It has been reported that zinc-finger transcription factor Slug plays a critical role in tumor proliferation and differentiation, relapse, invasion, metastasis, and decreased survival. However, there is little information on the expression and role of Slug in salivary adenoid cystic carcinoma (ACC). METHOD Demographic variables and primary tumor site, dates of diagnoses, perineural invasion, local regional recurrence, and distant metastasis of 121 cases of salivary ACC treated at the Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University between 1996 and 2005 were retrieved. The Slug expression of all these cases was examined by immunohistochemical methods. The association between Slug expression and clinicopathological variables was analyzed using Chi-squared test, and the prognostic factors were examined by univariate and multivariate analyses. RESULTS Our data demonstrated that the positive expression of Slug was observed in 71.90% (87/121) of cases. Slug expression was significantly associated with tumor site, TNM stage, histological pattern, perineural invasion, local regional recurrence and distant metastasis of patients with ACC (P<0.05). And Slug expression, local regional recurrence and distant metastasis were independent and significant prognostic factors in all patients. CONCLUSIONS It is proposed that Slug may play an important role in the invasion and metastasis of ACC, and that Slug had the potential for one of the strong invasion and metastasis indicators at presentation of ACC patients.