Longjiang Li

CXCR4 Promotes Oral Squamous Cell Carcinoma Migration and Invasion through Inducing Expression of MMP-9 and MMP-13 via the ERK Signaling Pathway

The increased migration and invasion of oral squamous cell carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. Although the chemokine receptor CXCR4 and its ligand, stromal cell–derived factor-1α, have been found to play an important role in tumor invasion, its precise role and potential underlying mechanisms remain largely unknown. In this study, we showed that knockdown of CXCR4 significantly decreased Tca8113 cells migration and invasion, accompanied with the reduction of MMP-9 and MMP-13 expression. Inhibition of ligand binding to CXCR4 by a specific antagonist TN14003, also led to reduced cancer cell migration and invasion. Because the degradation of the extracellular matrix and the basement membrane by proteases, such as matrix metalloproteinases (MMP) is critical for migration and invasion of cancer cells, we investigated the expression of several MMPs and found that the expression of functional MMP-9 and MMP-13 was selectively decreased in CXCR4 knockdown cells. More importantly, decreased cell migration and invasion of CXCR4 knockdown cells were completely rescued by exogenous expression of MMP-9 or MMP-13, indicating that the two MMPs are downstream targets of CXCR4-mediated signaling. Furthermore, we found the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in CXCR4-silenced cells, suggesting that ERK may be a potential mediator of CXCR4-regulated MMP-9 and MMP-13 expression in Tca8113 cells. Taken together, our results strongly suggest the underlying mechanism of CXCR4 promoting Tca8113 migration and invasion by regulating MMP-9 and MMP-13 expression perhaps via activation of the ERK signaling pathway. Mol Cancer Res; 9(2); 161–72. ©2011 AACR.

In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia.

Purpose: Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis. Experimental Design: We select p53(−) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia. Results: Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G1 in POE-9n cells by inducing p21CIP/WAF and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21CIP/WAF protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement. Conclusion: Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia. (Clin Cancer Res 2009;15(21):6724–31)

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.

RNAi targeting CXCR4 inhibits tumor growth through inducing cell cycle arrest and apoptosis.

CXC chemokine receptor 4 (CXCR4) is involved in many human malignant tumors and plays an important role in tumor growth and metastasis. To explore the effects of CXCR4 expression on the malignant cells of oral squamous cell carcinoma (OSCC), Tca8113 and SCC-9 cell lines, as well as their xenograft models, of nude mice were used to detect cancer cell proliferation alteration. This study also examined the corresponding molecular mechanism after CXCR4 knockdown using a recombinant lentiviral vector expressing small interference RNA (siRNA) for CXCR4. RNA interference-mediated knockdown of CXCR4 in highly aggressive (Tca8113 and SCC-9) tumor cells significantly inhibited the proliferation of the two cell lines in vitro and in vivo. The expression levels of >1,500 genes involved in cell cycle, apoptosis, and multiple signaling pathways were also altered. These results provide new evidence of CXCR4 as a promising tumor gene therapeutic target.

Phase I Study of Repeated Intraepithelial Delivery of Adenoviral p53 in Patients With Dysplastic Oral Leukoplakia

PURPOSE Advances in tumor biology and clinical trials indicate that p53 transfer is an alternative therapy for head and neck squamous cell carcinoma. The aim of this phase I clinical trial is to evaluate the feasibility, safety, and biologic activity of multiple intraepithelial injections of recombinant adenovirus (rAd)-p53 in patients with dysplastic oral leukoplakia (OLK), the most common precursor of the oral squamous cell carcinoma. PATIENTS AND METHODS Eighteen Chinese patients clinically and histopathologically diagnosed as having dysplastic OLK were recruited for this study. On a 15-day cycle, intraepithelial injections of rAd-p53 were administered once every 3 days at dose levels of 1 x 10(8) virus particles/cm(2). During treatment, patients were monitored for adverse events, and enzyme-linked immunosorbent assay was used to detect the serum antiadenoviral immunoglobulin (Ig) G/IgM. Incisional biopsies were performed 24 to 48 hours after the last injection, and immunohistochemistry was used to examine the protein expression of p53, p21, and bcl-2. The patients were followed up for 6 months to observe the initial clinical effect. RESULTS All 18 patients received a total cycle without dose-limiting toxicity, and administration was feasible and well tolerated. Adenovirus IgG/IgM turned from negative to positive after the 4 injections with rAd-p53. After treatment, p53 protein expression and p21 protein expression were significantly enhanced (100% and 89.9%, respectively), yet bcl-2 protein presented low expression (16.7%). During the treatment and follow-up, 13 patients (72.2%) showed a clinical response to treatment. CONCLUSIONS Intraepithelial injections of Gendicine (SiBiono GeneTech, Shenzhen, China) were safe, feasible, and biologically active for patients with dysplastic OLK. It may be a promising treatment for OLK.

Application of K/Sr co-doped calcium polyphosphate bioceramic as scaffolds for bone substitutes

Ion doping is one of the most important methods to modify the properties of bioceramics for better biodegrade abilities, biomechanical properties, and biocompatibilities. This paper presents a novel ion doping method applied in calcium polyphosphate (CPP)-based bioceramic scaffolds substituted by potassium and strontium ions (K/Sr) to form (K/Sr–CPP) scaffolds for bone tissue regeneration. The microstructure and crystallization of the scaffolds were detected by scanning electron microscopy and X-ray diffraction. Compressive strength and degradation tests were assessed to evaluate the mechanical and chemical stabilities of K/Sr–CPP in vitro. The cell biocompatibility was measured with respect to the cytotoxicity of the extractions of scaffolds. Muscle pouches and bone implantation were performed to evaluate the biodegradability and osteoconductivity of the scaffolds. The results indicated that the obtained K/Sr–CPP scaffolds had a single beta-CPP phase. The unit cell volume and average grain size increased but the crystallization decreased after the ions were doped into the CPP structure. The K/Sr–CPP scaffolds yielded a higher compressive strength and a better degradation property than the pure CPP scaffold. The MTT assay and in vivo results reveal that the K/Sr–CPP scaffolds exhibited a better cell biocompatibility and a tissue biocompatibility than CPP and hydroxyapatite scaffolds. This study proves the potential applications of K/Sr–CPP scaffolds in bone repair.

Chronic stress promotes oral cancer growth and angiogenesis with increased circulating catecholamine and glucocorticoid levels in a mouse model

OBJECTIVES Chronic stress was previously reported to play a role in the development of oral cancer, yet the correlation between stressors and oral cancer progression is not well understood. MATERIALS AND METHODS We implanted human oral cancer cell line CAL 27 in nude mice to investigate the effects of chronic stress on tumor growth, and designed a physical restraint system to create an experimentally stressed animal model, in which periodic immobilization induced characteristic chronic stress. Tumor burdened animal were randomly assigned into four groups: (a) control group, (b) daily stress for 2h with light, (c) daily stress for 2h in dark, and (d) daily stress for 6h with light. Animals were sacrificed after three weeks. Various analyses were performed on parameters including body weight, tumor weight, in situ expression of MMP-2 and VEGF, and the plasma concentrations of epinephrine, norepinephrine and glucocorticoid. RESULTS AND CONCLUSION Our data showed that chronic stress resulted in greater tumor size, more expression of MMP-2 and VEGF, higher level of plasma catecholamines, and more invasive growth of oral carcinoma cells in a mice model. We have successfully set up an animal model, which studied the effect of chronic stress on oral carcinoma growth rate and progression. These data further suggested that catecholamine and glucocorticoid might stimulate tumor progression under chronic stress.

High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma

It has been shown that interstitial fluid pressure (IFP) is elevated in many solid tumors. The elevated IFP in tumors is responsible, at least in part, for the poor blood supply, inadequate delivery of therapeutic agents to solid tumors and poor treatment response in patients. The present study was carried out to examine alterations in malignant phenotypes in oral squamous cell carcinoma cells subjected to conditions mimicking IFP and to identify the relevant molecular mechanisms. We investigated tumor cell proliferation and invasion using SCC-4 and SCC-9 cells subjected to an increased extracellular pressure of 0, 15 and 30 mmHg in vitro. The results revealed that the increased IFP resulted in a marked increase in cancer cell proliferation, survival and invasion in vitro and altered the expression of >1,800 genes involved in invasion and metastasis, the heat shock pathway, the p38 and JNK signaling pathway, apoptosis and the cell growth and differentiation signaling pathway. These results suggest the important potential clinical application of measuring IFP, which can be used as a generic marker of prognosis and response to therapy.

Progressively bilateral resorption of the mandible

We describe a case of arrested spontaneous mandibular resorption for which there was no effective treatment. A 32-year-old male patient presented to our department with mandibular resorption so severe that the residual mandibular body bone resembled chopsticks. A provisional diagnosis of Gorham-Stout Syndrome (GSS) was made. VEGF levels found in plasma and tissue fluid was believed to be a possible marker for the condition. Although there was no histological evidence for the diagnosis, a combination of the history, physical changes, and pathological findings in the soft tissues, we believe the diagnosis of GSS using VEGF as a marker was reasonable. There are over 200 reports of GSS in the literature. Most of the research has focused on the pathologic changes in the bone, but the condition of the related soft tissues has usually been ignored. We believe that this report is one of the first cases in which the mandibular resorption has been arrested for some time, during which the soft tissue is still in a pathological condition.

Microenvironment construction of strontium–calcium-based biomaterials for bone tissue regeneration: the equilibrium effect of calcium to strontium

Strontium-doped calcium phosphate-based biomaterials have gained increased recognition due to their beneficial effects on bone formation. However, the underlying mechanism is still not clear. In this study, we detected the calcification effects of strontium-based materials on osteoblasts in vitro and bone formation in vivo. The results showed that strontium may inhibit bone cell function in osteoblasts under a standard calcium concentration (1.8 mM) by both reducing alkaline phosphatase activity and inhibiting absorption of osteopontin and osteocalcin. In contrast, a high calcium concentration (9 mM) enhances the bone regeneration effect of strontium-based materials. Cultured osteoblasts underwent increased proliferation, calcification and alkaline phosphatase activity upon increasing calcium concentrations. An experimental animal model was utilized to simulate a high calcium concentration microenvironment in bone tissue and low calcium concentration in the subcutaneous part and the in vivo results are similar to the in vitro results. These findings suggest that strontium only promoted an anabolic effect on osteoblasts to enhance osteogenesis in a calcium rich microenvironment. Strontium would inhibit bone regeneration under a low dose of calcium in vivo. Therefore, strontium seems to be a potentially effective therapeutic option for bone regeneration in combination with a high concentration environment of calcium ions. These results would provide an in-depth knowledge of an ion-based bone tissue substitute for bone regeneration.