SCC7 Cells Research Articles

Self-assembled hyaluronic acid nanoparticles for active tumor targeting

Hyaluronic acid nanoparticles (HA-NPs), which are formed by the self-assembly of hydrophobically modified HA derivatives, were prepared to investigate their physicochemical characteristics and fates in tumor-bearing mice after systemic administration. The particle sizes of HA-NPs were controlled in the range of 237–424 nm by varying the degree of substitution of the hydrophobic moiety. When SCC7 cancer cells over-expressing CD44 (the receptor for HA) were treated with fluorescently labeled Cy5.5-HA-NPs, strong fluorescence signals were observed in the cytosol of these cells, suggesting efficient intracellular uptake of HA-NPs by receptor-mediated endocytosis. In contrast, no significant fluorescence signals were observed when Cy5.5-labeled HA-NPs were incubated with normal fibroblast cells (CV-1) or with excess free-HA treated SCC7 cells. Following systemic administration of Cy5.5-labeled HA-NPs with different particle sizes into a tumor-bearing mouse, their biodistribution was monitored as a function of time using a non-invasive near-infrared fluorescence imaging system. Irrespective of the particle size, significant amounts of HA-NPs circulated for two days in the bloodstream and were selectively accumulated into the tumor site. The smaller HA-NPs were able to reach the tumor site more effectively than larger HA-NPs. Interestingly, the concentration of HA-NPs in the tumor site was dramatically reduced when mice were pretreated with an excess of free-HA. These results imply that HA-NPs can accumulate into the tumor site by a combination of passive and active targeting mechanisms.

Growth inhibition and apoptosis by (−)-epicatechin gallate are mediated by cyclin D1 suppression in head and neck squamous carcinoma cells

In recent studies, green tea components have been shown to induce cell growth arrest and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. In this report, we have investigated the effects of epicatechin gallate (ECG), one of the catechins in green tea, on anti-cancer activity in vitro. We found that cyclin D1 was highly expressed in HNSCC cells, and ECG suppressed 90% of cyclin D1 expression in SCC7 cells. We have also evaluated the effect of ECG on cell growth and apoptosis, showing that ECG (50 μM) exhibited a significant inhibition (50%) on the growth of SCC7 cells via G1 cell cycle arrest. ECG suppressed cyclin D1 in SCC7 cells in a dose- and time-dependent manner, and the suppression of the β-catenin pathway by ECG is one of the mechanism to facilitate ECG-induced cell growth arrest. These results suggest that ECG has a potential usage as a chemopreventive agent in HNSCC.

Enhanced Nectin-1 Expression and Herpes Oncolytic Sensitivity in Highly Migratory and Invasive Carcinoma

Although a variety of malignant tumors are susceptible to therapy with oncolytic herpes simplex viruses, the determinants of tumor sensitivity to these viruses are poorly understood. Nectin-1 is a cell surface adhesion molecule that is a component of intercellular adherens junctions and also functions as a herpes viral receptor. Because highly invasive cells may have decreased intercellular adhesion, we sought to determine if such cells might also have altered availability of cell surface nectin-1 to act as a herpes receptor. A series of squamous cell carcinoma lines of increasing migratory and invasive potential, termed MG1-MG14, were selected by serial passages of murine SCC7 through Matrigel invasion chambers. Available cell surface nectin-1 was enhanced on the MG11 and MG14 cell lines in comparison to SCC7 as measured by cellular ELISA and immunofluorescence microscopy. A replication-competent, oncolytic herpes virus (NV1023) showed an increased ability to enter MG11 and MG14 cells as compared with SCC7 cells. Furthermore, MG11 and MG14 supported increased herpes viral replication and cytotoxicity over SCC7. For all three of the cell lines, viral entry assays revealed that the actively migrating cells were significantly more susceptible to herpes infection than the nonmigrating cells. These results show that malignant cells with highly migratory and invasive properties may exhibit increased cell surface nectin-1 availability, which may serve as a herpes viral receptor to enhance the efficacy of herpes oncolytic therapy. This finding has implications regarding patient selection for future clinical trials using these promising therapeutic vectors.

590. Efficient, Serum-Resistant Transfection of Murine Squamous Cell Carcinoma Cells by Metafectene and GeneJammer: Application to HSV-tk/Ganciclovir Gene Therapy

Current treatments of head and neck squamous cell carcinoma (HNSCC) are largely unsatisfactory, and the five-year survival rate has not improved over the last two decades. The genetic approach to the treatment of HNSCC is based on the hypothesis that expression of therapeutic genes in target cells will cause a cytotoxic effect or mediate apoptosis. Oral cancer is a particularly appropriate target for gene therapy, since direct injection of genes into most primary and recurrent lesions is possible. Although generally efficient in transducing cells, viral vectors suffer from problems of immunogenicity, toxicity, limits in the size of exogenous DNA, and the risk of inducing tumorigenic mutations and generating active viral particles through recombination. Synthetic cationic liposome-DNA (lipoplexes) or cationic polymer-DNA complexes (polyplexes) constitute a promising alternative to the use of viral vectors and provide a simple means of transferring DNA into target cells. The inhibitory effect of serum on transfection mediated by non-viral vectors represents a serious limitation for their use in vivo. The presence of serum proteins, however, is unavoidable in vivo, and is advantageous in vitro because it increases cell viability and reduces transfection-associated cytotoxicity. In this study, we examined the effect of high concentrations of fetal bovine serum (FBS) on the delivery of luciferase, |[szlig]|-galactosidase and Herpes Simplex Virus thymidine kinase (HSV-tk) gene to murine squamous cell carcinoma cells, SCC-7, by the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer. Transfection was optimized using a luciferase-expressing plasmid. The level of gene expression in cell lysates was evaluated by measuring enzyme activity after a 48-h incubation. The optimal ratios for delivering the luciferase gene were 2 |[mu]|l Metafectene:1 |[mu]|g DNA and 3 |[mu]|l GeneJammer:0.5 |[mu]|g DNA. When transfection was performed in the presence of 20-60% FBS, both Metafectene- and GeneJammer-mediated luciferase expression were reduced only by about 20%. SCC-7 cells were also transfected with the |[szlig]|-galactosidase expressing plasmid in the absence or presence of 60% FBS. Approximately 60-70% of the cells were positive for |[szlig]|-gal staining. The expression of |[szlig]|-galactosidase was essentially not affected by serum. Cells transfected with the HSV-tk plasmid were incubated in the absence or the presence of ganciclovir (GCV; 20 |[mu]|g/ml) for the indicated periods of time. The Alamar Blue assay was used to determine GCV-mediated cytotoxicity. Mock-transfected cells served as controls. The delivery of the HSV-tk gene by Metafectene in the absence and presence of 60% FBS, followed by GCV treatment for 3 days, resulted in 68% and 36% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 60% cytotoxicity. After 6 days, 90-100% cytotoxicity was observed in the presence of 0% or 60% FBS. Our observations suggest that both GeneJammer and Metafectene may be useful for the gene therapy of OSCC in animal models.

Potential role of caspase-3 and -9 in arsenic trioxide-mediated apoptosis in PCI-1 head and neck cancer cells

Arsenic trioxide (As2O3) has been shown to inhibit the proliferation of hematologic malignant cells. Previously, we reported that As2O3 had an antitumoral effect in head and neck cancer. Here, we investigated the induction of apoptosis and its mechanism in PCI-1 head and neck squamous carcinoma cells, after treatment with As2O3. Treatment with 2 microM of As2O3 caused apoptosis in PCI-1 cells following 3 days of exposure, which was detected by the annexin V-PI and DAPI staining methods. The cell death population was markedly increased, being 88% larger than the As2O3-untreated control cells. To address the mechanism of apoptosis, a Western blot assay was performed, showing that Bax was up-regulated without a change in Bcl-2. Activation of caspase-9 during As2O3-induced apoptosis was substantiated by monitoring the proteolysis of the caspase-9, which was associated with an increase of Apaf-1 and cytochrome c protein. PCI-1 cells rapidly changed the mitochondria membrane potential (DeltaPsim) after addition of As2O3. Furthermore, activation of caspase-3 was demonstrated by monitoring the proteolysis of the caspase-3 and by measuring caspase-3 activity with a fluorogenic substrate, which was associated with the cleavage of poly(ADP-ribose) polymerase. To examine the in vivo effect of As2O3, C3H mouse inoculated with syngenic SCC7 cells was treated by intratumoral injection of As2O3 (300 microg) every day, demonstrating that tumor mass was dramatically reduced on day 4, and revealed induction of apoptosis by TUNEL assay. These results suggest that apoptosis of PCI-1 cells by As2O3 is induced by activation of caspase-3 via cytochrome c, caspase-9 and Apaf-1 complex.

Radioresistance in Murine Solid Tumors Induced by Interleukin-1

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1 alpha on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1 alpha on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 after administration of IL-1 alpha (0.5 micrograms/mouse, ip) and characterized by increases in D(o), Dq, alpha/beta and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1 alpha had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1 alpha was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1 alpha had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D(o), alpha/beta and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1 alpha under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1 alpha in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1 alpha may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different.