Lai-ping Zhong

MicroRNAs contribute to the chemoresistance of cisplatin in tongue squamous cell carcinoma lines

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. They are strongly implicated in human cancers, including oral squamous cell carcinoma (OSCC). Evidence for the involvement of miRNAs as important regulators of chemosensitivity and chemoresistance in OSCC is not well understood. In this study, miRNA microarray was firstly used to compare the differential miRNAs levels between the cisplatin-sensitive tongue squamous cell carcinoma line (Tca8113) and its cisplatin-resistant subline (Tca/cisplatin). Three miRNAs of miR-21, -214, and -23a were validated by miRNAs real-time PCR, and intervened by anti-miRNA oligonucleotides (miR-214 and -23a) and pre-miRNA plasmid transfection (miR-21). Further relationship between miR-23a and DNA topoisomerase II beta (TOP2B) on the chemoresistance against cisplatin was studied. There were 19 out of 480 differential miRNAs between the Tca8113 and Tca/cisplatin cells. miR-214 and -23a were found increased as with chemoresistance against cisplatin in the Tca/cisplatin cells while miR-21 was found decreased as with chemosensitivity for cisplatin in the Tca/cisplatin cells. Intervention of these three miRNAs could decrease the chemoresistance against cisplatin in Tca/cisplatin cells. Transfection of anti-miR-23a into the Tca/cisplatin cells could increase the TOP2B protein expression. Our results suggest the existence of differential miRNAs with chemosensitivity and chemoresistance between the cisplatin-sensitive and resistant tongue squamous cell carcinoma lines. miR-21 serves as a chemosensitive miRNA, while miR-214 and -23a serve as chemoresistant miRNAs in the tongue squamous cell carcinoma lines. miR-23a is an up-stream regulator of TOP2B to realize the chemoresistance of cisplatin.

The NF-kappa B inhibitor, celastrol, could enhance the anti-cancer effect of gambogic acid on oral squamous cell carcinoma.

BackgroundGambogic acid (GA) is a major active ingredient of gamboge, a widely used traditional Chinese medicine that has been reported to be a potent cytotoxic agent against some malignant tumors. Many studies have shown that the NF-kappa B signaling pathway plays an important role in anti-apoptosis and the drug resistance of tumor cells during chemotherapy. In this study, the effects and mechanisms of GA and the NF-kappa B inhibitor celastrol on oral cancer cells were investigated.MethodsThree human oral squamous cell carcinoma cell lines, Tca8113, TSCC and NT, were treated with GA alone, celastrol alone or GA plus celastrol. Cytotoxicity was assessed by MTT assay. The rate of apoptosis was examined with annexin V/PI staining as well as transmission electronic microscopy in Tca8113 cells. The level of constitutive NF-kappa B activity in oral squamous cell carcinoma cell lines was determined by immunofluorescence assays and nuclear extracts and electrophoretic mobility shift assays (EMSAs) in vitro. To further investigate the role of NF-kappa B activity in GA and celastrol treatment in oral squamous cell carcinoma, we used the dominant negative mutant SR-IκBα to inhibit NF-kappa B activity and to observe its influence on the effect of GA.ResultsThe results showed that GA could inhibit the proliferation and induce the apoptosis of the oral squamous cell carcinoma cell lines and that the NF-kappa B pathway was simultaneously activated by GA treatment. The minimal cytotoxic dose of celastrol was able to effectively suppress the GA-induced NF-kappa B pathway activation. Following the combined treatment with GA and the minimal cytotoxic dose of celastrol or the dominant negative mutant SR-IκBα, proliferation was significantly inhibited, and the apoptotic rate of Tca8113 cells was significantly increased.ConclusionThe combination of GA and celastrol has a synergistic antitumor effect. The effect can be primarily attributed to apoptosis induced by a decrease in NF-kappa B pathway activation. The NF-kappa B signaling pathway plays an important role in this process. Therefore, combining GA and celastrol may be a promising modality for treating oral squamous cell carcinoma.

Overexpression of cytokeratin 17 protein in oral squamous cell carcinoma in vitro and in vivo

OBJECTIVE To determine the cytokeratin 17 (CK17) expression in oral squamous cell carcinoma (OSCC) both in vitro and in vivo. METHODS Comparative proteomic analysis of an in vitro cellular carcinogenesis model of OSCC (including a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells and another kind of cells (HB56 cells) at the early stage of carcinogenesis was performed to identify differentially expressed proteins. CK17 was further validated in vitro (cellular carcinogenesis model and other three OSCC lines) and in vivo (tissues from six healthy persons and 30 primary OSCC patients) by Western blotting and immunohistochemistry respectively. RESULTS Increased CK17 expression was identified by two-dimensional gel electrophoresis and liquid chromatography-tandem mass chromatography in the HB56 and HB96 cells over HIOECs. Western blotting confirmed the increased CK17 expression in the HB56, HB96 cells and other three OSCC lines. Immunohistochemistry confirmed the increased CK17 expression in the cancerous tissues from OSCC patients compared with the paired adjacent non-malignant epithelia. CONCLUSION Increased CK17 expression may play an important role in the carcinogenesis progression of OSCC; however, further studies on the molecular function of CK17 are encouraged to clear the precise mechanism of CK17 in OSCC.

Characteristics of a cancerous cell line, HIOEC-B(a)P-96, induced by benzo(a)pyrene from human immortalized oral epithelial cell line.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. In vitro study on OSCC cell lines, especially derived from immortalized oral epithelial cells, is a very useful strategy to understand the mechanism of carcinogenesis. Based on our previous human immortalized oral epithelial cell (HIOEC) line, obtained from normal oral epithelial cells by transfection of HPV16 E6/E7 gene, a new cancerous cell line, HIOEC-B(a)P-96 (HB96), was established from the HIOEC by induction with benzo(a)pyrene. The characteristics of the HB96 cells such as cell morphology, ultrastructure, proliferation ability, invasion ability, and tumorigenesis were studied. The HB96 cells lost contact inhibition with uncontrolled cell division and obvious cell overlap, they were polygonal in shape and ununiform in size with increased ratio between nucleus and plasma. Increased proliferative ability and invasion ability were confirmed by the cell proliferation analysis and cell invasion assay, respectively. The tumorigenicity of well to moderately differentiated squamous cell carcinoma was confirmed in the nude mice experiments pathologically. Increased expression of HPV16 E6/E7 proteins and obvious correlation with decreased expression of p53 and Rb proteins was also confirmed by Western blotting. Thus, this HB96 cell line induced by benzo(a)pyrene from the HIOEC line is a useful tool to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses. It is also the first in vitro cancerous cell line of OSCC in China derived from immortalized oral epithelial cells.

Study of induction chemotherapy efficacy in oral squamous cell carcinoma using pseudotargeted metabolomics.

The effect of induction chemotherapy on oral cancer is controversial owing to inconsistent results. However, the efficacy of induction chemotherapy is closely related to locoregional recurrence, distant metastasis, and overall survival after the treatment. A pseudotargeted metabolomics revealed that metabolites involved in glycolysis and amino acid metabolism were inversely regulated in patients with different chemotherapy responses, and most fatty acids, steroids, and antioxidant substances were up-regulated in all patients after the treatment. Among the metabolites, lactic acid, glucose, glutamic acid, aspartic acid, leucine, and glycerol were remarkably associated with induction chemotherapy efficacy. Subsequently, lactic acid, glutamic acid, and aspartic acid were defined as potential biomarkers of the suitability and efficacy of induction chemotherapy. Our results show that 100.0 and 84.37% of patients with different chemotherapy efficacy were correctly identified in the training and test sets, respectively. Moreover, patient suitability for treatment was correctly predicted for 100.0, 81.25, and 100.0% of patients in the training, test, and external validation sets, respectively. In conclusion, metabolites related to glycolysis, redox homeostasis, and anabolic progress were indicative of induction chemotherapy efficacy both pre- and post-chemotherapy and beneficial for outcome evaluation and prediction. These results illustrate the potentials of metabolomics in personalized induction chemotherapy.

Expression of growth differentiation factor 15 is positively correlated with histopathological malignant grade and in vitro cell proliferation in oral squamous cell carcinoma

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) and expression microarray analysis showed that the gene encoding growth differentiation factor 15 (GDF15) was significantly upregulated in this model. In this study, we confirmed that expression of GDF15 was increased both at mRNA and protein levels in a panel of OSCC lines and clinical samples from primary OSCC patients. We also observed that expression of GDF15 was positively correlated with the malignancy of the disease: a higher level of GDF15 expression indicates a higher malignant grade of OSCC. Treatment of OSCC cell line (Tca3118) with siRNA against GDF15 significantly inhibited cellular proliferation and colony formation. Based on these observations, we conclude that GDF15 is a positive gene of OSCC development and progression and GDF15 can be used as an additional marker for histopathologic evaluation of OSCC differentiation.

Association between IFN-α and primary Sjogren's syndrome

OBJECTIVE To determine the level of IFN-alpha in labial salivary glands, plasma, and peripheral blood cells from patients with primary Sjogrens syndrome (pSS). METHODS Labial salivary gland biopsy specimens, plasma, and peripheral blood cells from patients with pSS were investigated. The IFN-alpha-positive cells, measurable IFN-alpha level, and IFN-alpha gene mRNA level were determined by using immunohistochemistry, ELISA, and real-time PCR, respectively. Statistical analysis was performed by Student t test or Fishers exact test. RESULTS About 60% of patients (22/37) with pSS had significantly higher scores of IFN-alpha-positive cells in labial gland biopsy and most IFN-alpha-positive cells were localized predominantly in the lymphocytes and ductal epithelial cells. But in 3 of the control samples (3/24), the IFN-alpha-positive cells existed only in the ductal epithelial cells with lower scores. Forty-three percents of the patients with pSS were found with detectable IFN-alpha concentration in plasma (> or = 12.5 pg/mL), and their concentration was higher than that of control group. Furthermore, the IFN-alpha mRNA levels in peripheral blood cells were up-regulated in the patients with pSS. CONCLUSION No matter in labial salivary glands or hematoplasma, or peripheral blood cells, IFN-alpha expression levels are up-regulated in patients with primary Sjogrens syndrome.

Decreased expression of Annexin A1 correlates with pathologic differentiation grade in oral squamous cell carcinoma

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB96 cells. In this study, comparative proteomic analysis identified that Annexin A1 was one of the significantly down-regulated genes in the cancerous HB96 cells. To investigate Annexin A1 down-regulation and its potential usefulness as a molecular marker in OSCC, we further screened Annexin A1 expressions with a panel of OSCC lines, and clinical samples of cancerous and the paired adjacent normal tissues from primary OSCC patients. By Western blot analysis and real-time PCR, we showed that both Annexin A1 mRNA and protein expressions decreased in OSCC cell lines except in two cell lines for the mRNA levels. Immunohistochemistry and real-time PCR also showed that both Annexin A1 mRNA and protein expressions decreased in the cancerous tissues from OSCC patients compared with those in the paired adjacent non-malignant epithelia. More importantly, both Annexin A1 mRNA and protein expressions negatively correlated with the pathologic differentiation grades of cancerous tissues. The lower Annexin A1 mRNA or protein expressions correlated with the poorer pathologic differentiation grades. These results suggest that decreased expression of Annexin A1 contributes to the cancerous progression of OSCC, and Annexin A1 may be a potential biomarker for pathologic differentiation grade of OSCC.

Induction chemotherapy in patients with resectable head and neck squamous cell carcinoma: a meta-analysis.

BackgroundInduction chemotherapy has been investigated as a possible strategy to shrink or downstage locally advanced head and neck cancers, providing opportunity to remove the lesions completely after induction chemotherapy, especially in the patients with resectable advanced disease. The aim of this study was to investigate the definitive effect of induction chemotherapy in patients with resectable head and neck squamous cell carcinoma.MethodsA meta-analysis of randomized trials (1965–2011) was performed on the impact of induction chemotherapy on survival, disease control, and toxicity in this population of patients. Kaplan-Meier curves were read by Engauge-Digitizer. Data combining was performed using RevMan.ResultsFourteen trials (2099 patients) were involved in this analysis. There was no significant difference on overall survival, disease free survival, or locoregional recurrence between the patients treated with and without induction chemotherapy (P >0.05). However, the patients treated with induction chemotherapy had a lower rate of distant metastasis by 8% (95% confidence interval 1%–16%, P = 0.02) than those treated without induction chemotherapy. In patients with laryngeal cancer, comparing to radical surgery, the larynx could be preserved in responders to induction chemotherapy without survival decease (P >0.05). Induction chemotherapy-associated death was 0%–5%.ConclusionsBased on the results above, there is a significant benefit of induction chemotherapy on decreasing distant metastasis in patients with resectable head and neck squamous cell carcinoma. In patients with laryngeal cancer, induction chemotherapy provides larynx preservation in responders to induction chemotherapy.

Adenoid cystic carcinoma of the head and neck: clinicopathologic analysis of 218 cases in a Chinese population.

OBJECTIVE The aim of this study was to analyze the clinicopathologic characteristics and prognostic factors of adenoid cystic carcinoma of the head and neck (ACCHN). STUDY DESIGN This was a retrospective study of 218 patients with ACCHN. RESULTS The cohort included 110 men and 108 women; the parotid and the palate were the most common site of involvement. Of 203 patients with follow-up information (range 2-132 months), 57 had died of the tumor. Distant metastasis (DM) and local recurrence (LR) were documented in 83 (40.9%) and 34 (16.7%) patients, respectively. Cox regression analysis indicated that a solid pattern was a marker for LR and that positive margins and older age were risk factors for DM. Histologic pattern, T stage, N stage, LR, DM, and patient age contributed to the prediction of disease-specific survival. CONCLUSIONS A solid pattern, metastasis, LR, and older age are the most important factors for predicting poor prognosis in Chinese patients with ACCHN.