Yun Hong

Expressions of CXCL12/CXCR4 in oral premalignant and malignant lesions.

Objective. The chemokine receptor CXCR4 and its ligand CXCL12 have been suggested to play important roles in the initiation or progression of cancers. The goal of the present study was to investigate alterations of CXCL12/CXCR4 in oral premalignant lesions and oral squamous cell carcinoma (OSCC). Methods. In 13 normal oral epithelia, 24 dysplastic oral leukoplakia (OLK), and 40 OSCC specimens, expressions of CXCL12 and CXCR4 were evaluated by immunohistochemistry. Results. CXCR4 was expressed in 37.5% of OLK and 60% of OSCC. CXCL12 was detected in 50% of OLK and 62.5% of OSCC. In OLK, CXCR4 positive ratio showed no significant difference from normal epithelia, but the CXCL12 positive ratio was significantly higher. Significant relationship between CXCL12 and CXCR4 was found both in OLK and OSCC. Conclusion. Our results indicated that CXCL12/CXCR4 axis may play roles from early steps of oral malignant transformation and contribute to the progress of oral carcinogenesis.

Increased expression of focal adhesion kinase correlates with cellular proliferation and apoptosis during 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis

BACKGROUND Oral carcinogenesis is a multistep process and requires accumulation and interplay of a series of molecular genetic events. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in signalling pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. Overexpression of FAK has been linked to oral squamous cell carcinoma (OSCC). So, it is hypothesized that FAK expression might contribute to oral carcinogenesis. METHODS During 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis, FAK protein expression, proliferating cell nuclear antigen (PCNA) and apoptotic nuclei (TUNEL assay) were examined by means of immunohistochemistry. RESULTS Along with the progress of multistage carcinogenesis, FAK expression increased significantly among different histopathological groups with normal mucosa, mild-dysplastic epithelia, moderate-dysplastic epithelia, severe-dysplastic epithelia and in turn OSCC. Furthermore, FAK immunohistochemical index and PCNA-labelling index displayed positive correlation (r = 0.946, P < 0.05), while negative associations were revealed between apoptotic index and final FAK index (r = -0.959, P < 0.05). CONCLUSION Our results implicated a role for FAK in oral carcinogenesis. Inhibition of FAK might be a potential novel treatment strategy in this disease.

Frequent mutation of p16CDKN2A exon 1 during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide

In this study we explored the mutation types of p16CDKN2A exon 1 and the corresponding frequencies in experimental rat tongue carcinogenesis. Twenty barrier Sprague–Dawley (SD) rats were divided into the control (n = 5) and experimental group (n = 15), to which 4‐nitroquinoline‐1‐oxide (4‐NQO) in drinking water was administered. Two samples of normal, three samples of moderate/severe dysplasia and four samples of invasive squamous cell carcinoma lesions were selected following strict histopathological examination in double‐blind manner. The PCR products of p16CDKN2A exon 1 amplified from these tissues were sequenced. Point mutations of p16CDKN2A exon 1 were found in all of the precancerous and cancerous lesions. Half of the mutations were detected on guanine (G). Twenty mutations, including a missense mutation of the start codon resulting in alternative reading frame of p16CDKN2A exon 1, were also identified. These preliminary results suggested that mutation of p16CDKN2A exon 1 might be an early molecular event of rat tongue carcinogenesis induced by 4NQO and G was the mutation hotspot.

The In Vitro and In Vivo Antitumor Effects of Clotrimazole on Oral Squamous Cell Carcinoma

Background Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC. Methodology In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole. Results Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model. Conclusion Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

All-trans retinoic acid restores gap junctional intercellular communication between oral cancer cells with upregulation of Cx32 and Cx43 expressions in vitro

Objective: All-trans retinoic acid (ATRA) has been demonstrated to inhibit tumor growth by restoration of gap junctional intercellular communication (GJIC) via upregulation of connexin (Cx) expression in some solid tumors. However, the relationship between ATRA and GJIC remains unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the effect of ATRA on the GJIC function of OSCC. Study design: We measured the effects of ATRA on the viability and cell cycle distribution of SCC9 and Tca8113 OSCC cells. The GJIC function was observed using the scrape-loading dye transfer technique, and the mRNA and protein levels of Cx32 and Cx43 were detected by qRT-PCR, Western blot, and immunofluorescence assays. Results: ATRA inhibited the growth of OSCC cells in a dose- and time-dependent manner (P <0.05) and caused cell cycle arrest. ATRA-treated cells showed a 2.69-fold and 2.06-fold enhancement of GJIC in SCC9 and Tca8113 cells, respectively (P <0.05). Moreover, ATRA induced upregulation of Cx32 and Cx43 at both the mRNA and protein levels in OSCC cells. Conclusion: Our results indicated that restoration of GJIC via enhanced Cx32 and Cx43 expression might serve as a novel mechanism for the anti-tumor effect of ATRA in OSCC. Key words:All-trans retinoic acid, oral squamous cell carcinoma, connexin, gap junctional intercellular communication.

Elevated autocrine chemokine ligand 18 expression promotes oral cancer cell growth and invasion via Akt activation

Chemokine (C-C motif) ligand 18 (CCL18) has been implicated in the pathogenesis and progression of various cancers; however, in oral squamous cell carcinoma (OSCC), the role of CCL18 is unknown. In this study, we found that CCL18 was overexpressed in primary OSCC tissues and was associated with an advanced clinical stage. CCL18 was found in both the cytoplasm and cell membrane of OSCC cells and was predominantly produced by cancer epithelial cells, as opposed to tumor-infiltrating macrophages. In vitro studies indicated that the effects of endogenous CCL18 on OSCC cell growth, migration, and invasion could be blocked by treatment with a neutralizing anti-CCL18 antibody or CCL18 knockdown, while exogenous recombinant CCL18 (rCCL18) rescued those effects. Akt was activated in rCCL18-treated OSCC cells, while LY294002, a pan-PI3K inhibitor, abolished both endogenous and exogenous CCL18-induced OSCC cell invasion. In vivo, LY294002 treatment attenuated rCCL18-induced OSCC cell growth. Our results indicate that CCL18 acts in an autocrine manner via Akt activation to stimulate OSCC cell growth and invasion during OSCC progression. They also provide a potential therapeutic target for the treatment of oral cancer.

Modulation of IL-1β reprogrammes the tumor microenvironment to interrupt oral carcinogenesis.

Head and neck squamous cell carcinoma (HNSCC) development is a multistage process includes the normal, dysplasia and squamous cell carcinoma (SCC) stages. Recently, increasing evidence has suggested that the tumor microenvironment (TME) is an integral part of malignant transformation. Exploring certain key node genes in TME for future intervention in dysplasia to interrupt oral carcinogenesis was the primary goal of this research. To achieve this goal, systems biology approaches were first applied to the epithelia and fibroblasts collected at sequential stages in a 4-nitroquinoline-1-oxide (4NQO) - induced rat oral carcinogenesis model. Through bioinformatics network construction, IL-1β was identified as one of the key node genes in TME during carcinogenesis. Immunohistochemical staining of human and rat samples demonstrated that IL-1β expression patterns were parallel to the stages of malignant transformation. Silencing IL-1β with lentivirus-delivered shRNA significantly inhibited oral squamous cell carcinoma cell growth both in vivo and in vitro. Based on these findings, we hypothesized that IL-1β may be a chemoprevention target in TME during oral carcinogenesis. Therefore, we targeted IL-1 in the TME by oral mucosal injection of an IL-1 receptor antagonist in 4NQO rats. The results demonstrated that targeting IL-1 could interrupt oral carcinogenesis by reprogramming the TME.

p16CDKN2A expression during rat tongue carcinogenesis induced by 4-Nitroquinoline-1-oxide

p16(CDKN2A) is one of the most important tumor-suppressor genes and has been investigated widely in recent years for its role in oral carcinogenesis, but few have explored the relationship between its RNA and protein, especially in precancerous tissues. The aim of this study was to explore the relationship of mRNA and protein level of p16(CDKN2A) in rat tongue carcinogenesis process induced by 4-nitroquinoline-1-oxide. By the use of semi-quantitative RT-PCR, immunohistochemistry (IHC) and Western Blot, histologically normal, premalignant and invasive squamous cell carcinoma samples from the animal model were explored respectively. The results showed the levels of mRNA of p16(CDKN2A) did not significantly change during the carcinogenesis process when compared with controls. However, detectable level of P16 protein expression was lost in both the dysplasia and carcinoma groups. We could conclude that p16(CDKN2A) in 4NQO-induced rat tongue carcinogenesis might be inactivated predominantly by posttranscriptional regulation.

IL-1β maintains the redox balance by regulating glutaredoxin 1 expression during oral carcinogenesis.

BACKGROUND Interleukin-1 beta (IL-1β) is a pleiotropic cancer-inflammation-linked cytokine which has been reported upregulated in many cancers. In our previous study, IL-1β was found to be one of the key node genes during oral malignant transformation, and glutaredoxin 1 (Grx1) was identified as one of the downstream genes of IL-1β in tumor microenvironment. Grx1 is ubiquitous oxidoreductase which is necessary for scavenging reactive oxygen species (ROS) and the intracellular redox balance maintenance. METHODS Tissues from different stages of mucosal malignant transformation were obtained from 4NQO-induced rat oral carcinogenesis model and human mucosa for Grx1 expression detection by immunohistochemical staining. The intracellular ROS levels and Grx1 mRNA level of oral squamous carcinoma cell CAL27 were detected after IL-1β treatment with or without pretreatment of IL-1Ra or NAC, respectively. The ROS levels were detected in Leti-si-IL-1β and Leti-si-NC CAL27 cells after IL-1β stimulation. The invasion and migration abilities of CAL27 cells were tested by transwell assay after IL-1β stimulation with or without pretreatment of IL-1Ra. RESULTS Grx1 expression was associated with the malignant transformation process in vivo. Exogenous IL-1β upregulated the intracellular ROS level and the expression of Grx1 in CAL27 cells, which could be counteracted by IL-1Ra. The intracellular ROS accumulation induced by exogenous IL-1β was responsible for the Grx1 upregulation. Endogenous IL-1β acted as a switch in regulating the ROS level by modulating Grx1 expression, which was involved in the invasion and migration of OSCC cells. CONCLUSIONS IL-1β finely orchestrated the redox balance during carcinogenesis by modulating Grx1 expression.

Stromal-epithelial lactate shuttle induced by tumor‑derived interleukin‑1β promotes cell proliferation in oral squamous cell carcinoma

Stromal-epithelial lactate shuttle is an essential process to support fast-growing tumor cells, however, the underlying mechanism remains ambiguous. Interleukin-1β (IL-1β), which is a key node gene in both stromal and epithelial cells of oral squamous cell carcinoma (OSCC), may participate in this metabolic reprogramming. In the present study, anaerobic glycolysis of cancer-associated fibroblasts (CAFs) was evaluated and the role of IL-1β in regulating stromal-epithelial lactate shuttle was determined. A co-culture system of primary fibroblasts and OSCC cell lines (CAL27, UM1 or SCC25) was created to investigate the stromal-epithelial interaction. α-smooth muscle actin (α-SMA) expression of fibroblasts, IL-1β expression and cell proliferation of OSCC cells, and a series of glycolytic genes were measured. Recombinant IL-1β treatment and IL-1β knockdown in UM1 cells were also used to evaluate the effect of IL-1β. Expression of α-SMA, glucose transporter 1, hexokinase 2, lactic dehydrogenase and mono-carboxylate transporter (MCT) 4 were significantly overexpressed in activated fibroblasts, while IL-1β and MCT1 were upregulated in OSCC cells, indicating enhanced glycolysis in cells of the tumor stroma and a lactate shuttle to the tumor cells. Furthermore, exogenous IL-1β induced fibroblasts to present similar expression profiles as that in the co-culture system. Silencing of IL-1β significantly abrogated the regulatory effect of UM1 cells on stromal glycolysis. Additionally, carboxy-fluorescein succinimidyl ester cell tracing indicated that OSCC cell proliferation was accelerated during co-cultivation with fibroblasts. These results indicate that tumor-derived IL-1β enhanced stromal glycolysis and induced one-way lactate flow from the tumor mesenchyme to transformed epithelium, which promotes OSCC proliferation.