Mouse Squamous Carcinoma Cells Research Articles

Synthesis and Cytostatic Effect of 3'-deoxy-3'-C-Sulfanylmethyl Nucleoside Derivatives with d-xylo Configuration.

A small library of 3’-deoxy-C3’-substituted xylofuranosyl-pyrimidine nucleoside analogues were prepared by photoinduced thiol-ene addition of various thiols, including normal and branched alkyl-, 2-hydroxyethyl, benzyl-, and sugar thiols, to 3’-exomethylene derivatives of 2’,5’-di-O-tert-butyldimethylsilyl-protected ribothymidine and uridine. The bioactivity of these derivatives was studied on tumorous SCC (mouse squamous carcinoma cell) and immortalized control HaCaT (human keratinocyte) cell lines. Several alkyl-substituted analogues elicited promising cytostatic activity in low micromolar concentrations with a slight selectivity toward tumor cells. Near-infrared live-cell imaging revealed SCC tumor cell-specific mitotic blockade via genotoxicity of analogue 10, bearing an n-butyl side chain. This analogue essentially affects the chromatin structure of SCC tumor cells, inducing a condensed nuclear material and micronuclei as also supported by fluorescent microscopy. The results highlight that thiol-ene chemistry represents an efficient strategy to discover novel nucleoside analogues with non-natural sugar structures as anticancer agents.

Combining anticancer agents photodynamic therapy and LCL85 leads to distinct changes in the sphingolipid profile, autophagy, caspase-3 activation in the absence of cell death, and long-term sensitization

Two anticancer agents, LCL85 and photodynamic therapy (PDT) were combined to test whether the combination PDT/LCL85 evokes changes in the sphingolipid (SL) profile and promotes cell death. Treatment of SCCVII mouse squamous carcinoma cells using the silicone phthalocyanine Pc 4 for PDT induced increases in the prodeath global ceramides/dihydroceramides (DHceramides), and no changes in the prosurvival sphingosine-1-phosphate (S1P). In contrast, after LCL85, the levels of most ceramides and DHceramides were reduced, whereas the levels of S1P were increased. After PDT/LCL85 the levels of global ceramides and DHceramides, and of S1P, were restored to resting levels. PDT/LCL85 also enhanced the levels of C18-, C20-, and C20:1-ceramide, and C18-DHceramide. Treatment with PDT, with or without LCL85, led to substantial reductions in sphingosine levels. PDT/LCL85 induced enhanced autophagy and caspase-3 activation. None of the treatments affected short-term viability of cells. In contrast, long-term clonogenic survival was reduced not only after PDT or LCL85, but even more after PDT/LCL85. Overall, our data show that short-term exposure to PDT/LCL85 led to distinct signature effects on the SL profile, enhanced autophagy, and caspase-3 activation without cell death. Long-term exposure to PDT/LCL85 enhanced overall cell killing, supporting translational potential of PDT/LCL85.

C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing

Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT), an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30) was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing.

Abstract 585: Treatment of squamous cell carcinoma with ultrasound and microbubble mediated gene therapy

Abstract Background: Ultrasound contrast agents are gas-filled microbubbles (MB) that can be induced to vibrate or cavitate in an ultrasound field. When the MB are loaded with genes and systemically injected, ultrasound-targeted MB destruction (UTMD) has been shown to facilitate focused delivery and transduction of genes to the target site. An experimental model of mouse squamous cell carcinoma was used to test the hypothesis that delivery of a suicide gene via UTMD would slow tumor growth. Methods: Lipid based perfluorobutane gas-filled MBs (2 μm diameter) were synthesized incorporating a cationic phospholipid to enable loading with DNA (100 ug per 109 MB). DNA loading was confirmed using electrophoresis and DNAseI challenge. Primary cultured mouse squamous carcinoma cells were subcutaneously injected into mice. 3-5 days after cell injection, mice were intravenously infused over 30 min with MB (5 × 108) loaded with plasmids containing reporter genes GFP (n = 3) or luciferase (n = 6). During MB infusion, ultrasound (1.3MHz) was intermittently delivered using a clinical scanner at high acoustic power, with simultaneous ultrasound imaging used to confirm MB destruction within the tumor. To assess the therapeutic potential of the system, tumors were allowed to grow to 100 µl, and MB (5 × 108) loaded with plasmid containing either the suicide gene thymidine kinase (TK) (n = 6) or GFP control (n = 6) was given to separate groups of mice along with ultrasound treatment. Gancyclovir was administered 3 days after UTMD and tumors were serially measured until they were 2ml in volume, after which animals were euthanized and tissue harvested for histology. Results: UTMD-mediated delivery of reporter genes resulted in tumor expression of luciferase (1568 RLU/min/mg) and GFP (<1%) that exceeded expression in control tumors treated with microbubbles + plasmid only (p = 0.02). GFP expression was seen in both peri-vascular areas as well as individual tumor cells, both in small percentages. The doubling rate of TK-treated tumors (2.91 days) was lower than that of GFP treated tumors (3.44 days, p = 0.02) despite equal ultrasound and gancyclovir treatment regimes. Relative to GFP-treated tumors, TK-treated tumors had greater apoptosis (p = 0.04) and increased microvascular density (p = 0.03). Conclusions: Our data demonstrate the principle that the acoustic behavior of MB can be exploited to therapeutic end. Single dose UTMD-mediated delivery of suicide gene slows the growth of squamous cell carcinoma in this experimental model. Tumor growth suppression could be through an anti-angiogenic mechanism, as reporter gene expression was largely peri-vascular in location. Increased microvasculature seen in TK-treated tumors might be a result of compensatory angiogenesis, although this requires further study. UTMD is a promising non-viral method for targeting gene therapy which may be useful in the treatment of a spectrum of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 585.

425 The role of p27 in efficacy of chemo- and radiosensitivity of primary mouse squamous carcinoma cells

Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs.In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP.It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance.

Laminin-5 promotes adhesion and migration of epithelial cells: identification of a migration-related element in the gamma2 chain gene (LAMC2) with activity in transgenic mice.

The effects of laminin-5 and its subunit gamma2 chain on cell adhesion and migration were studied, and a migration-related cis-acting element was identified in the gamma2 chain gene (LAMC2) using promoter-reporter gene constructs in transgenic mice. Intact laminin-5 molecules, but not recombinant gamma2 chain promoted cell adhesion of human keratinocytes and mouse squamous carcinoma cells, indicating that the gamma2 chain does not contain a cellular binding site. However, the gamma2 chain as such is probably involved in the process of cell locomotion, as antibodies against the short arm of the chain inhibited migration of carcinoma cells in an in vitro assay. Further evidence for the involvement of the gamma2 chain in cell migration was obtained by the identification of a cis-acting element in a promoter-lacZ reporter gene construct that was active in migratory epithelial cells of healing wounds in mice made transgenic by microinjection of the construct into fertilized oozytes. The migration active element was located in the sequence between -613 and +55. The same construct, and another one containing 5900 base pairs of the 5' flanking region, yielded very limited expression in cells of normal tissues. The limited expression was, however, only observed in epithelial cells of different tissues, i.e. cell types that normally express laminin-5 in vivo. The results show that the sequence between -613 and +55 contains elements that can drive expression during epithelial cell migration and that also partially confers more general epithelium expression. However, elements outside -5900 and +55 are needed for normal epithelium expression of the LAMC2 gene.

Immunobiology of lipid-modulated UV-carcinogenesis

Previous studies have demonstrated that high levels of dietary fat exacerbate UV-carcinogenic expression and suppress immunoresponsiveness. The latter may account for the former response. We have explored this possibility through T-lymphocyte transfer studies. Groups of HRA.HRII-c/ + /Skh hairless mice were fed isocaloric diets containing high (12%, wt./wt.) or low (0.75%) levels of corn oil and irradiated 5 days/week (1.0 J cm −2/day) for 11 weeks with filtered FS-40 sunlamps. At weeks nine and 12, enriched T-cells from high-fat donors that had received 11 weeks of UV were transferred intravenously to low-fat recipients. Median tumor times for high-fat, low-fat recipient, and low-fat groups were 15.8, 18.5, and 21.6 weeks, respectively. The significantly ( P<0.03) shortened primary tumor latent period in low-fatfed animals resulting from transfer of relatively low levels of T-cells derived from chronically irradiated high-fat donors demonstrates that the influence of dietary fat upon UV-carcinogenic expression is, at least partially, mediated via immunologic mechanisms. Further studies suggest that fat-modulated carcinogenesis can, itself, be regulated immunologically. A soluble T-14 (mouse squamous carcinoma cell line) cell-free fraction was injected subcutaneously at axillae and inguen of animals fed the high-fat diet during the first three weeks of UV or immediately post-UV. At week four post-UV, animals were challenged with T-14 cells injected subcutaneously at both flanks. 21 days post-challenge the tumor volumes of low-fat and high-fat immunized animals were zero versus 593 mm 3 for the high-fat group ( P < 0.007). Such treatment significantly ( P< 0.03) increases the latent period of UV-induced primary tumors as well, when compared to non-treated high-fatfed animals.

Assay system for tumor control effect using encapsulated multicellular spheroid models

Most advanced head and neck cancers are inoperable, hence they are treated by either radiotherapy or combined chemoradiotherapy. More effective methods of combined chemoradiotherapy are certainly required for improving the curability of these cancers. Numerous factors involved in modifying the effectiveness have to be quantitatively analyzed for improvement; this has been a limiting factor preventing therapeutic progress. The purpose of the present study was to establish an experimental technique for assessing the in vivo effect of radiotherapy or chemoradiotherapy on multicellular tumor spheroids encapsulated in a semitransparent microcapsule. Mouse squamous carcinoma cells suspended in sodium alginate solution were transferred to a syringe connected to an air jet apparatus. Droplets containing cells were dropped into a calcium chloride solution, which caused them to gel, and then treated with poly L-lysine solution. The encapsulated cells were cultured in vitro for three weeks to form multicellular spheroids with central necrosis. The spheroids were either X-irradiated in vitro or in vivo by grafting in a nude mouse. The dose-cell survival curve in vivo was biphasic with a radioresistant component which was not observed in vitro. This method was considered to be applicable to human tumor cells and useful for determining the effectiveness of therapeutic agent as human solid tumor models in vivo.

Regulation of major acute-phase plasma proteins by hepatocyte-stimulating factors of human squamous carcinoma cells.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.