Pharyngeal Carcinoma Cells Research Articles

Abstract B168: Dietary polyphenols inhibit pepsin and prevent airway epithelial cell hyperproliferation associated with laryngeal cancer.

Abstract A number of otolaryngologic inflammatory and neoplastic diseases are associated with gastric reflux extending proximal to the esophagus. Extraesophageal reflux, also known as laryngopharyngeal reflux (LPR), increases the risk of developing laryngeal cancer by approximately two-fold and analyses of neoplastic development in patients with prior gastrectomy suggest that nonacid components of refluxate promote laryngeal carcinogenesis as well. Clinical and basic science research implicates gastric pepsin as a key deleterious agent of LPR. Our previous research demonstrates that pepsin at pH7 induces hyperproliferation and inflammatory and cell cycle regulatory gene expression changes in airway epithelial cells in vitro. Acid suppression therapy through proton-pump inhibitors (PPI's), the most commonly prescribed therapeutic for reflux, has demonstrated minimal efficacy in resolution of LPR-attributed disease. Phytochemicals derived from algae, pine, and most recently Noni fruit, however, have been shown to not only attenuate reflux-attributed lesions and inflammation in rat models of disease but also directly inhibit peptic activity. This study examines the potential of plant polyphenols curcumin, ecabet sodium (ES) and an anthocyanin-enriched fraction of freeze-dried black raspberry (BRB) to inhibit peptic activity and pepsin-mediated cell damage and proliferative changes in cultured human laryngeal and pharyngeal cells. Cultured human laryngeal epithelial cells from punch biopsy, pharyngeal carcinoma (FaDu) and gastric adenocarcinoma (AGS) cells were pre-treated with/without curcumin (10uM), ES (125ug/ml) and BRB (100ug/ml) for 30 minutes, rinsed and subsequently treated with/without nonacid pepsin (0.1mg/ml, pH7.0) for 1 hour. Morphological changes were examined by transmission electron microscopy and proliferative changes by cell count assay and realtime PCR array (curcumin only). Direct inhibition of peptic activity was determined by in vitro kinetic assay. Curcumin, ES and BRB significantly inhibited peptic activity (p<0.05) in a dose-dependent manner at uM or ug/ml concentrations. Curcumin, ES and BRB abrogated mitochondrial damage and significantly reduced hyperproliferation elicited by pepsin in human laryngeal and pharyngeal cells. Curcumin attenuated 28 of the 31 changes in cell cycle regulatory gene expression induced by pepsin in pharyngeal cells. Gastric cells, which would not be expected to exhibit sensitivity to pepsin-mediated damage, displayed neither mitochondrial damage nor hyperproliferation following treatment with pepsin. These data demonstrate that plant polyphenols curcumin, ES and BRB are protective against pepsin-mediated airway epithelial cell damage and hyperplasia and implicate dietary modification/supplementation with plant polyphenols as a novel pepsin-protective therapeutic modality for diseases associated with proximal reflux including laryngeal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B168.

Down-Regulation of EBV-LMP1 Radio-Sensitizes Nasal Pharyngeal Carcinoma Cells via NF-κB Regulated ATM Expression

BackgroundThe latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice.ResultsIn this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells.ConclusionsTogether, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.

Polyinosine‐polycytidylic acid enhances cellular adherence of Streptococcus pneumoniae

Viral upper respiratory tract infections (URIs) are often followed by secondary bacterial infections. To better understand this phenomenon, we examined the impact of the viral agent polyinosine-polycytidylic acid [Poly (I:C)] on the adherence of Streptococcus pneumoniae (Spn) to pharyngeal epithelial cells. In vitro model of cultured human pharyngeal epithelial cells. Detroit 562 cells, a human pharyngeal carcinoma cell line, were pretreated with Poly (I:C). Poly (I:C)-induced expression of platelet-activating factor receptor (PAF-R) was assayed using real-time polymerase chain reaction, flow cytometry, and immunofluorescence microscopy. Bacterial adhesion to these epithelial cells was assessed using immunofluorescence microscopy and colony formation assays. Pretreatment with Poly (I:C) increased mRNA and protein expression of PAF-R in Detroit 562 cells and enhanced the adherence of Spn to these epithelial cells. RNA viral infection can enhance PAF-R expression in epithelial cells and increase the adherence of Spn. These findings might explain in part the mechanisms that underlie the increase in bacterial infection following URIs.

Tumour Cell Lines HT-29 and FaDu Produce Proinflammatory Cytokines and Activate Neutrophils In Vitro: Possible Applications for Neutrophil-Based Antitumour Treatment

There is evidence that polymorphonuclear neutrophils (PMNs) can exert severe antineoplastic effects. Cross-talk between tumour cells and endothelial cells (ECs) is necessary for the accumulation of PMN around a tumour. This work reports the ability of two PMN-sensitive, human, permanent cell lines—colorectal adenocarcinoma (HT-29) and pharyngeal squamous-cell carcinoma (FaDu) cells—to act as inflammatory foci. PMNs were cytotoxic to both lines, the adhesion of the PMNs to the tumour cells being important in this effect. The tumour cells released appreciable amounts of IL-8 and GROα, and induced the transmigration of PMN through human microvascular-EC monolayers. Conditioning media associated with both lines induced the adhesion of PMN and the surface expression of ICAM-1 in microvascular-EC. In addition, FaDu-conditioning-medium strongly induced the production of proinflammatory cytokines by microvascular-EC. These results support the idea that tumour cells might normally induce a potent acute inflammatory response, leading to their own destruction.

Antibody-Mediated Blockade of Integrin αvβ6 Inhibits Tumor Progression In vivo by a Transforming Growth Factor-β–Regulated Mechanism

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Lack of effect of palifermin on the anti-tumor activity of cisplatin, cetuximab or panitumumab in the FaDu human head and neck carcinoma model

6060 Background: The FaDu human pharyngeal carcinoma cell line expresses the receptors for both KGF and EGF. Palifermin is a recombinant form of human KGF. The objective of this study was to determine whether the combination of palifermin with cisplatin and the EGFR inhibitors cetuximab or panitumumab interferes with the anti-tumor activities of these agents in the FaDu head and neck xenograft model. Methods: FaDu tumor cells were implanted subcutaneously in CD1 nude mice. For the combination of palifermin with cetuximab and cisplatin, treatment began on day 9 and for the combination of palifermin with panitumumab and cisplatin, treatment began on day 10. The primary endpoint was the measure of tumor volume. Results: There was no statistically significant difference in tumor volume between the control arm and palifermin alone (p = 0.9993, 0.9831). Neither was there a statistically significant difference between the combination of palifermin, cisplatin and cetuximab compared to the combination of cisplatin and cetuximab (p = 0.7730). Similarly, there was no significant difference between the combination of palifermin, cisplatin and panitumumab compared to the combination of cisplatin and panitumumab (p = 0.9739). Conclusions: In drug combination settings, the addition of palifermin did not affect the therapeutic efficacy of cisplatin, cetuximab or panitumumab in any of the two- or three-way drug combinations. When used alone, palifermin did not promote the growth of the FaDu human xenograft despite the fact that these tumors express KGFR. The lack of interference of palifermin with the anti-tumor activity of cisplatin and the anti-EGFR target therapeutics suggests it is appropriate to move forward clinically investigating the use of palifermin in solid tumor settings in which these therapeutic modalities are used. No significant financial relationships to disclose.

Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells

Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. Hypoxia-induced CA IX protein expression was seen after >6 h at < or =5% O(2), and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1alpha. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro and can be applied to sort chronically hypoxic cells from tumors.

Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1α (HIF-1α)

Hypoxia-inducible factor-1α (HIF-1α) is both a potential endogenous marker of tumor hypoxia and therapeutic target, and elevated HIF-1α protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cells in vitro. Here, we found that in FaDu xenografts, no significant HIF-1α protein accumulation was detectable by either flow cytometry or Western blot, despite the presence of hypoxic (pimonidazole-positive, radiation resistant) cells. To investigate the effect of different tumor microenvironment conditions on hypoxic HIF-1α accumulation, we performed in vitro hypoxia experiments (0.1% O 2, 24 h) with manipulation of pH (7.4 vs. 6.7), glucose (0–5.5 mM) and serum (0 or 10%) availability in FaDu and HT 1080 human fibrosarcoma cells. Hypoxic induction of HIF-1α protein was strongly dependent on glucose availability and largely abolished at 0.55 mM glucose or less in both cell lines. This glucose effect was confirmed in a hypoxia-responsive-element (HRE)/enhanced-green-fluorescent-protein (EGFP) reporter assay in transfected HT 1080 cells and possibly explains a lack of HIF-1α protein in hypoxic tumor cells.

Low-Level Doxorubicin Resistance in Benzo[a]pyrene-Treated KB-3-1 Cells Is Associated with Increased LRP Expression and Altered Subcellular Drug Distribution

The P-glycoprotein (P-gp)-negative epidermoid pharyngeal carcinoma cells KB-3-1 were grown in 0.25 mM benzo[a]pyrene (BaP) for 3 months and increased resistance to doxorubicin, but not to vinblastine, colchicine, or cisplatin, was found. Doxorubicin resistance was not altered by cyclosporin, the P-gp inhibitor. Intracellular accumulation of BaP or calcein, a substrate for P-gp and multidrug resistance protein (MRP), was not altered by inhibitors of the P-gp and MRP. The expression of cytochrome P450 (CYP) 1A1, lung-resistance-related protein (LRP), P-gp, and MRP was investigated. Overexpression of CYP1A and LRP, on the mRNA and protein levels, was found. BaP-treated KB-3-1 cells remained P-gp negative while the level of MRP was not altered. Subcellular accumulation of BaP was found to be localized in the cytoplasm and minimal in the nuclei in BaP treated cells. In contrast, even penetration of BaP to the nuclei and cytoplasm was found in untreated cells. Subcellular distribution of doxorubicin was altered following BaP treatment with localized accumulation of the cancer drug in cytoplasmic organelles but not in the nuclei. Our data suggested that LRP might play a protective role against toxic compounds. The correlation of increased expression of LRP, but not P-gp nor MRP, with decreased doxorubicin accumulation in the nuclear target suggests a pivotal role of this perinuclear transporter in the MDR phenotype of P-gp-negative cancer cells. These results also propose an alternative mechanism of cancer drug resistance emergence, namely, induction of LRP activity following treatment with BaP, an environmental toxicant and a carcinogen.

The effect of culture conditions on the in-vitro adherence of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant isolates of Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behaviour into two subgroups, epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA) strains. An existing adherence assay was modified to determine differences in adherence properties between these two groups of MRSA, and the influence of culture conditions on the adherence of SMRSA and EMRSA strains to plastic, human collagen I (HuCol I) and pharyngeal carcinoma Detroit 562 cells (D562) was determined. In-vitro parameters, such as culture medium, growth temperature and growth phase of the bacterium, influenced the adherence of MRSA strains to plastic significantly. Even more pronounced differences in adherence due to changes in growth conditions and growth phase of the bacteria were found for the adherence of MRSA strains to HuCol I. Growth phase had a significant effect on the adherence of MRSA strains to the pharyngeal carcinoma cells D562. However, the study did not find conditions which made it possible to distinguish EMRSA from SMRSA strains. These data show that extrapolation of in-vitro data concerning adherence of MRSA strains to in-vivo conditions should be treated with caution.

Enhancement of sensitivity to CDDP in human pharyngeal carcinoma cells by H-7, a protein kinase inhibitor

We investigated the synergetic effects of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) on cis-diamminedichloroplatinum(II) (CDDP) cytotoxicity to KB cells, a human pharyngeal carcinoma cell line, and explored the possible mechanism whereby it exerted these effects. In vitro treatment with H-7 markedly decreased protein kinase C (PKC) activity in KB cells and significantly sensitized the cells to CDDP. Treatment of KB cells with H-7 induced a dose-dependent depletion of intracellular glutathione (GSH) levels. These results suggest that the decreased GSH content induced by H-7 may be involved in sensitizing KB cells to CDDP.