Ning Ji

Involvement of potential pathways in malignant transformation from Oral Leukoplakia to Oral Squamous Cell Carcinoma revealed by proteomic analysis

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common forms of cancer associated with the presence of precancerous oral leukoplakia. Given the poor prognosis associated with oral leukoplakia, and the difficulties in distinguishing it from cancer lesions, there is an urgent need to elucidate the molecular determinants and critical signal pathways underlying the malignant transformation of precancerous to cancerous tissue, and thus to identify novel diagnostic and therapeutic target.ResultsWe have utilized two dimensional electrophoresis (2-DE) followed by ESI-Q-TOF-LC-MS/MS to identify proteins differentially expressed in six pairs of oral leukoplakia tissues with dysplasia and oral squamous cancer tissues, each pair was collected from a single patient. Approximately 85 differentially and constantly expressed proteins (> two-fold change, P < 0.05) were identified, including 52 up-regulated and 33 down-regulated. Gene ontological methods were employed to identify the biological processes that were over-represented in this carcinogenic stage. Biological networks were also constructed to reveal the potential links between those protein candidates. Among them, three homologs of proteosome activator PA28 a, b and g were shown to have up-regulated mRNA levels in OSCC cells relative to oral keratinocytes.ConclusionVarying levels of differentially expressed proteins were possibly involved in the malignant transformation of oral leukoplakia. Their expression levels, bioprocess, and interaction networks were analyzed using a bioinformatics approach. This study shows that the three homologs of PA28 may play an important role in malignant transformation and is an example of a systematic biology study, in which functional proteomics were constructed to help to elucidate mechanistic aspects and potential involvement of proteins. Our results provide new insights into the pathogenesis of oral cancer. These differentially expressed proteins may have utility as useful candidate markers of OSCC.

Honokiol: a promising small molecular weight natural agent for the growth inhibition of oral squamous cell carcinoma cells

Honokiol (HNK) is a small organic molecule purified from magnolia species and has demonstrated anticancer activities in a variety of cancer cell lines; however, its effect on oral squamous cell carcinoma (OSCC) cells is unknown. We investigated the antitumor activities of HNK on OSCC cells in vitro for the first time. The inhibitory effects of HNK on the growth and proliferation of OSCC cells were demonstrated via in vitro 3‐(4,5‐dimethyl thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and propidium iodide (PI) assays, and the apoptotic cells were investigated by the observation of morphological changes and detection of DNA fragmentation via PI, TdT‐mediated dUTP‐biotin nick end labeling (TUNEL), and DNA ladder assays, as well as flow cytometry assay. The results showed that HNK inhibited the growth and proliferation of OSCC cells in vitro in a time and dose‐dependent manner. The inhibitory effect was associated with the cell apoptosis induced by HNK, evidenced by the morphological features of apoptotic cells, TUNEL‐positive cells and a degradation of chromosomal DNA into small internucleosomal fragments. The study also demonstrated here that the inhibition or apoptosis mediated by 15 μg·mL−1 or 20 μg·mL−1 of HNK were more stronger compared with those of 20 μg·mL−1 5‐fluorouracil (5‐Fu, the control) applied to OSCC cells, when the ratio of OSCC cell numbers were measured between the treatment of different concentrations of HNK to the 5‐Fu treatment for 48 h. HNK is a promising compound that can be potentially used as a novel treatment agent for human OSCC.

Oral cancer overexpressed 1 (ORAOV1) regulates cell cycle and apoptosis in cervical cancer HeLa cells.

BackgroundOral Cancer Overexpressed 1 (ORAOV1) is a candidate protooncogene locating on 11q13. Recent studies show that ORAOV1 acts as a primary driving force behind 11q13 gene amplification and plays a functional role in the tumorigenesis in a variety of human squamous cell carcinomas (SCCs). According to the results of molecular cytogenetic methods, 11q13 was characterized to be a high-level and recurrent amplification chromosomal site in cervical cancers. Up till now, the role of ORAOV1 in cervical cancer is unknown. The purpose of this study is to elucidate the function of ORAOV1 in cervical cancer cell growth by studying its roles in HeLa cells using small interfering RNA.ResultsFunctional analyses revealed that ORAOV1 was involved in the regulation of HeLa cell growth through its effect on cell cycle and apoptosis. Silence of ORAOV1 in HeLa cells downregulated the expression of Cyclin A, Cyclin B1 and Cdc2, and led to a distinct S cell cycle arrest. Moreover, knockdown of ORAOV1 expression activated both extrinsic and intrinsic apoptotic pathways and led to apoptosis in HeLa cells through its effect on the expression of several apoptosis related proteins such as P53, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and cytochrome c. Interestingly, the expression of Cyclin D1, a pivotal gene for cervical cancer tumorigenesis, was also found to be reduced in ORAOV1 silenced HeLa cells.ConclusionOur findings indicate that ORAOV1 has an important role in regulating cell growth of cervical cancer HeLa cells through regulating the cell cycle and apoptosis. Thus, it may be a crucial protooncogene and a novel candidate therapeutic target for cervical cancer.

Receptor for activated C kinase 1 (RACK1): a regulator for migration and invasion in oral squamous cell carcinoma cells

PurposeReceptor of activated protein kinase C 1 (RACK1) has been identified as an anchoring or adaptor protein in multiple intracellular signal transduction pathways. Our previous study has showed that the expression of RACK1 was paralleled with proliferation and correlated with metastasis and clinical outcome. However, the underlined mechanism has not been uncovered.Materials and methodsWe first selected a most effective siRNA among three siRNAs (siRNA-1, siRNA-2 and siRNA-3) targeting different regions in the RACK1 mRNA and re-evaluated the anticancer effect of RACK1 silencing on HSC-3 and Cal-27 cell lines by cell growth inhibition. And then, we investigated whether knockdown of RACK1 could inhibit cell adhesion, migration and invasion in these two cell lines. To further understand the molecular mechanism of RACK1 in these processes, the expressions of EGFR, pEGFR, HER2, MMP-2 and MMP-9 were detected by western blot.ResultsWe verified that the silence of RACK1 gene in two OSCC cell lines could not only inhibit cell proliferation but also decrease the invasion, migration and adhesion capability of the tumor cells. Further, western blot analysis deduced that it might be related to the decrease in protein expression of EGFR, pEGFR, HER2, MMP-2 and MMP-9.ConclusionOur results clearly showed the significance of RACK1-induced OSCC cell migration, invasion and adhesion, which could explain the underlined mechanism of the effect of the gene on metastasis and clinical outcome. Also, our results confirmed its role to be a prognostic indicator and a promising drug target for OSCC cell metastasis.

Biodegradable Thermosensitive Hydrogel for SAHA and DDP Delivery: Therapeutic Effects on Oral Squamous Cell Carcinoma Xenografts

Background OSCC is one of the most common malignancies and numerous clinical agents currently applied in combinative chemotherapy. Here we reported a novel therapeutic strategy, SAHA and DDP-loaded PECE (SAHA-DDP/PECE), can improve the therapeutic effects of intratumorally chemotherapy on OSCC cell xenografts. Objective/Purpose The objective of this study was to evaluate the therapeutic efficacy of the SAHA-DDP/PECE in situ controlled drug delivery system on OSCC cell xenografts. Methods A biodegradable and thermosensitive hydrogel was successfully developed to load SAHA and DDP. Tumor-beared mice were intratumorally administered with SAHA-DDP/PECE at 50 mg/kg (SAHA) +2 mg/kg (DDP) in 100 ul PECE hydrogel every two weeks, SAHA-DDP at 50 mg/kg(SAHA) +2 mg/kg(DDP) in NS, 2 mg/kg DDP solution, 50 mg/kg SAHA solution, equal volume of PECE hydrogel, or equal volume of NS on the same schedule, respectively. The antineoplastic actions of SAHA and DDP alone and in combination were evaluated using the determination of tumor volume, immunohistochemistry, western blot, and TUNEL analysis. Results The hydrogel system was a free-flowing sol at 10°C, become gel at body temperature, and could sustain more than 14 days in situ. SAHA-DDP/PECE was subsequently injected into tumor OSCC tumor-beared mice. The results demonstrated that such a strategy as this allows the carrier system to show a sustained release of SAHA and DDP in vivo, and could improved therapeutic effects compared with a simple additive therapeutic effect of SAHA and DDP on mouse model. Conclusions Our research indicated that the novel SAHA-DDP/PECE system based on biodegradable PECE copolymer enhanced the therapeutic effects and could diminished the side effects of SAHA/DDP. The present work might be of great importance to the further exploration of the potential application of SAHA/DDP-hydrogel controlled drug release system in the treatment of OSCC.

CAL 27 is an oral adenosquamous carcinoma cell line

CAL 27 is one of the most frequently used cell lines in the field of oral squamous cell carcinoma (OSCC) studying. However, our recent studies showed that the growth pattern of CAL 27 xenograft did not seem like typical OSCC. In this study, we verified the identity of CAL 27 cells by using by short tandem repeat analysis. Then, we performed tumor formation assay, HE staining and immunohistochemistry assay to further study the growing characteristics and histopathological diagnosis of CAL 27 xenografts. Our results showed that CAL 27 xenografts grew slowly in vivo with vesicle formation at both the surfaces and deeper areas of the tumors. The CAL 27 xenografts were then diagnosed as oral adenosquamous carcinomas. Thus CAL 27 appears to be an oral adenosquamous carcinoma cell line.

Cysteine dioxygenase type 1 promotes adipogenesis via interaction with peroxisome proliferator-activated receptor gamma.

Mammalian cysteine dioxygenase type 1 (CDO1) is an essential enzyme for taurine biosynthesis and the biodegradation of toxic cysteine. As previously suggested, Cdo1 may be a marker of liposarcoma progression and adipogenic differentiation, but the role of Cdo1 in adipogenesis has yet been reported. In this study, we found that the expression of Cdo1 is dramatically elevated during adipogenic differentiation of 3T3-L1 pre-adipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs). Conversely, knockdown of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation in 3T3-L1 cells and mBMSCs. Mechanistically, we found Cdo1 interacted with Pparγ in response to adipogenic stimulus. Further, depletion of Cdo1 reduced the recruitment of Pparγ to the promoters of C/EBPα and Fabp4. Collectively, our finding indicates that Cdo1 may be a co-activator of Pparγ in adipogenesis, and may contribute to the development of disease associated with excessive adipose tissue.

Human Beta-Defensin-1 Suppresses Tumor Migration and Invasion and Is an Independent Predictor for Survival of Oral Squamous Cell Carcinoma Patients

Background Human beta-defensin-1 (hBD-1) has recently been considered as a candidate tumor suppressor in renal and prostate cancer. The aim of this study was to investigate the role of hBD-1 in the progression of oral squamous cell carcinoma (OSCC) and its potential as diagnostic/prognostic biomarker and therapeutic target for OSCC. Methods HBD-1 expression in tissues at different stages of oral carcinogenesis, as well as OSCC cell lines was examined. HBD-1 was overexpressed in HSC-3, UM1, SCC-9 and SCC-25 cells and subjected to cell growth, apoptosis, migration and invasion assays. Tissue microarray constructed with tissues from 175 patients was used to examine clinicopathological significance of hBD-1 expression in OSCC. Results HBD-1 expression decreased from oral precancerous lesions to OSCC and was lower in OSCC with lymph node metastasis than those without metastasis. In vitro, the expression of hBD-1 was related to the invasive potential of OSCC cell lines. Induction of exogenous expression of hBD-1 inhibited migration and invasion of OSCC cells, probably by regulation of RhoA, RhoC and MMP-2; but had no significant effect on proliferation or apoptosis. In a cohort of patients with primary OSCC, cases with no expression of hBD-1 had more chance to be involved in lymph node metastasis. Eventually, the positive expression of hBD-1 was associated with longer survival of patients with OSCC, and multivariate analysis and ROC curve analysis confirmed hBD-1 positivity to be an independent prognostic factor of OSCC, especially OSCC at early stage. Conclusions Overall, these data indicated that hBD-1 suppressed tumor migration and invasion of OSCC and was likely to be a prognostic biomarker and a potential target for treatment of OSCC.

Self-Assembling Monomeric Nucleoside Molecular Nanoparticles Loaded with 5-FU Enhancing Therapeutic Efficacy against Oral Cancer

Conventional oligonucleotide based drug delivery systems suffer from lengthy synthetic protocols, high cost, and poor chemical or enzymatic stability under certain circumstances. Canonical free individual nucleosides cannot form stable nanostructures in aqueous solution as drug vehicles. Here, we report the development of a monomeric self-assembled nucleoside nanoparticle (SNNP) into an efficient drug delivery system which has currently no parallel in such field. This was achieved using a l-configurational pyrimido[4,5-d]pyrimidine nucleoside building block that can form robust discrete nanoparticles in just one step with water as the sole solvent. Its high biocompatibility and low toxicity was demonstrated in vitro and in vivo. In mouse xenograft model of oral squamous cell carcinoma (OSCC), SNNP loaded with 5-fluoro-uracile (5-FU-SNNP) remarkably retarded the tumor growth compared with free 5-FU, albeit SNNP alone showed no antitumor effect. The stability in blood circulation and the effective concentration of 5-FU in tumor tissue were increased upon the loading with SNNP. TUNEL and immunohistochemistry analyses further indicated that the superior in vivo antitumor efficacy of 5-FU-SNNP compared to free 5-FU was associated with an enhanced degree of inhibition of cell proliferation and stimulation of cell apoptosis. Furthermore, SNNP alleviated the toxic side effects of 5-FU. These findings suggested that when loaded with SNNP, 5-FU has better antitumor efficacy and lower side effects, indicating that SNNP can efficiently act as a readily accessible, robust, biocompatible and low-toxic nanobiomaterial which may find wide therapeutic applications clinically in the future.

Associations between proteasomal activator PA28γ and outcome of oral squamous cell carcinoma: Evidence from cohort studies and functional analyses.

Summary Background PA28γ was suggested to play a role in malignant progression. This paper aimed to investigate the association between PA28γ and the prognosis of oral squamous cell carcinoma (OSCC) in cohort studies. Methods The PA28γ expression level was assessed by immunohistochemistry in a total of 368 OSCC patients from three independent cohorts. The Cox proportional hazards regression model was used to determine multivariate hazard ratios for Overall Survival (OS). Model discrimination was measured using C Statistic. Additionally, OS was analyzed in Head Neck Squamous Cell Carcinoma (HNSCC) patients from The Cancer Genome Atlas (TCGA) data set. Functional analyses were conducted both in-vitro and in-vivo. Findings The median follow-up times of patients in the three studies were 60, 52, and 51 months. High expression of PA28γ was identified in tumors from 179 of 368 patients (48.6%). Compared with low expression, high expression of PA28γ was strongly associated with worse OS, with relative risks of 5.14 (95% CI, 2.51–10.5; P < 0.001), 2.82 (95% CI, 1.73–4.61; P < 0.001), and 3.85 (95% CI, 1.59–9.37; P = 0.003). PA28γ expression was also associated with disease-free survival in all three cohorts (P < 0.005). These findings are consistent with TCGA HNSCC data (P < 0.006). The prediction of all-cause mortality was significantly improved when PA28γ was added to the traditional clinical factors (Model 3, C statistic value: 0.78 VS 0.73, P = 0.016). In functional analyses, we found that PA28γ silencing dramatically inhibited the growth, proliferation and mobility of OSCC cells in vitro and reduced tumor growth and angiogenesis in tumor-bearing mice. Interpretation PA28γ overexpression is associated with adverse prognosis in patients with OSCC. The aberrant expression of PA28γ may contribute to the pathogenesis and progression of OSCC. Research in context OSCC is one of the most common HNSCC, which have a high lethally rate. However, few prognostic markers have been applied in the clinical practice. We found that PA28γ in OSCC tumor tissues were significantly high expression than those in normal tissues. As the results of the three cohorts from two independent research centers and from an additional validation cohort from a US population in the TCGA dataset, we demonstrate PA28γ is a good predictor of the risk of death in OSCC. Meanwhile, we demonstrate PA28γ have a potential role in OSCC tumorigenesis.