Human Pharyngeal Carcinoma Research Articles

Apoptotic effects of hyperoside on FaDu human pharyngeal carcinoma cells

Apoptotic effects of hyperoside on FaDu human pharyngeal carcinoma cells

A combination of all-trans retinoic acid derivative and COX-2 inhibitor has anticancer effects in human pharyngeal carcinoma cells

Backgrounds and aimsCarcinogenesis is characterized by an unlimited growth of cells exacerbated by Cox-2 overexpression. Cox-2 inhibitors have been proven effective in preventing and treating tumors. In our previous studies, we found that 4-Amino-2-Trifluoromethylphenyl Retinate (ATPR) induces cell apoptosis and inhibits cell proliferation to exhibit anti-cancer properties. The use of ATRA as well as Cox-2 inhibitors in clinical settings can cause adverse reactions. It is unknown what the effects and mechanisms of co-administration of ATPR and Cox-2 inhibitors are. ResultsA combination of ATPR and Cox-2 inhibitors, Celecoxib, inhibited pharyngeal cancer cell proliferation in vitro and induced apoptosis. The cell cycle was arrested at G0/G1 by activating P53 and CDNA1. By activating MAPK/JNK pathways, ATPR and Celecoxib led to intrinsic and extrinsic apoptosis in pharyngeal cancer cells. ATPR/Celecoxib combined treatment suppressed tumor growth in the pharyngeal cancer cell-derived xenograft mouse model by increasing the number of apoptotic cells. The expression of the RARA and PTGS2 genes was significantly increased in tumor tissue compared to non-tumor tissue in the clinical analysis of the head and neck squamous cell carcinoma dataset. An association was found between this and the level of intrinsic apoptotic signals. Furthermore, a survival analysis conducted over a period of five years indicated that higher levels of RARA expression were associated with a better clinical outcome. ConclusionATPR and celecoxib inhibit the proliferation of cancer cells as well as induce apoptosis. Co-administration of ATPR and Cox-2 inhibitors has the potential to be a novel treatment plan for cancer.

COMPOSITION ANALYSIS OF E-LIQUIDS AND THEIR EFFECTS ON HEALTHY LIVER AND PHARYNGEAL CARCINOMA CELL LINES

Electronic cigarettes have become popular worldwide in recent years although their effects on human health are still not properly known. The lack of regulations brings a problem of inconsistency between ingredients and the product label. We aimed to analyse the contents of widely used e-liquids and their effects on two different cell lines. Eleven e-liquid samples were selected according to their availability and popularity. Nicotine, propylene glycol (PG), glycerine (GLY), and volatile compounds in e-liquids were analysed by High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was used to determine the effects of e-liquids on transformed human normal liver epithelial cell line (THLE-2) and human pharyngeal carcinoma cell line (Detroit 562). Nicotine amounts were found to be consistent with product labels. and GLY were not only different between brands but also for products within the same brand. THLE-2 cell viability was inversely correlated with e-liquid concentration. However, decreases in cell viability were not correlated with nicotine amount. Interestingly, effects of several samples on Detroit 562 cells were triphasic; decrease in viability at lower doses, cell survival in mid-concentrations and loss of viability in highest doses. The analytical composition of e-liquids differs greatly among products which corresponds to different cellular effects. Viability of cancer cells does not change in a dose-dependent manner, which suggest that cellular differences may play role in the outcome of these products.

T52 attenuates oncogenic STAT3 signaling and suppresses osteosarcoma

BackgroundT52 is a steroidal saponin extracted from the traditional Chinese herb Rohdea fargesii (Baill.), and it is reported to possess strong anti-proliferative capabilities in human pharyngeal carcinoma cell lines. However, whether T52 has anti-osteosarcoma properties, and its potential mechanism is remains unknown. PurposeTo examine the outcome and underlying mechanism of T52 in osteosarcomas (OS). Methods/Study designsThe physiological roles of T52 in OS cells were examined using CCK-8, colony formation (CF), EdU staining, cell cycle/apoptosis and cell migration/invasion assays. The relevant T52 targets against OS were assessed via bioinformatics prediction, and the binding sites were analyzed by molecular docking. Western blot analysis was carried out to examine the levels of factors associated with apoptosis, cell cycle, and STAT3 signaling pathway activation. ResultsT52 markedly diminished the proliferation, migration, and invasion of OS cells, and promoted G2/M arrest and apoptosis in a dose-dependent fashion (DDF) in vitro. Mechanistically, molecular docking predicted that T52 stably associated with STAT3 Src homology 2 (SH2) domain residues. Western blot revealed that T52 suppressed the STAT3 signaling pathway, as well as the expression of the downstream targets, such as, Bcl-2, Cyclin D1, and c-Myc. In addition, the anti-OS property of T52 were partially reversed by STAT3 reactivation, which confirmed that STAT3 signaling is critical for regulating the anti-OS property of T52. ConclusionWe firstly demonstrated that T52 possessed strong anti-osteosarcoma property in vitro, which was brought on by the inhibition of the STAT3 signaling pathway. Our findings provided pharmacological support for treating OS with T52.

In Vitro Inhibition of Growth and Induction of Apoptosis by Mosinone-A in HEp-2 Oral Cancer Cells

The present study evaluated the influence of a Mosinone-A on the induction of apoptosis in HEp-2 human pharyngeal carcinoma cell lines as evidenced by cytotoxicity morphological changes, inhibition of cell proliferation in a time and dose dependent manner. Oral cancer is one of the leading causes of cancer worldwide. Many chemotherapeutics from plants have been tested in cancer, such as Mosinone-A, isolated from the bark of Annona squamous, acetogenin. further we analyzed anticancer activity by MTT assay, the generation of reactive oxygen species (ROS), the level of mitochondrial membrane potential and apoptotic morphological changes were analyzed by AO/EtBr. The results of an MTT assay demonstrated that Mosinone-A inhibited the growth of HEp2 cells in a dose-dependent manner. In addition, the induction of apoptosis was detected, as determined by morphological observation. These results indicated that Mosinone-A may induce apoptosis of HEp2 cells through the mitochondrial pathway.

Bactericidal effect of lascufloxacin on HEp-2 cell-internalized group A Streptococcus

IntroductionAlthough amoxicillin (AMPC) is recommended as first-line therapy for acute pharyngotonsillitis caused by group A streptococci (GAS), it often fails to eradicate infections. Internalization and subsequent intracellular survival of GAS are considered major mechanisms for penicillin therapeutic failure. It is, therefore, desirable to administer drugs that exert bactericidal effects on extracellular and intracellular GAS. In this study, we aim to investigate the bactericidal effects of lascufloxacin (LSFX) on internalized GAS in HEp-2 cells. Materials and methodsThe GAS strain M1 and clinical isolate strain #2 were used in this study. Following treatment of GAS-infected human pharyngeal carcinoma epithelial HEp-2 cells with LSFX or AMPC, internalized GAS cells were recovered. The concentrations of LSFX and AMPC were equivalent to 1 × and 2 × MIC for strain M1. Culture medium was used as a control. Time-lapse and fluorescence images of GAS invading HEp-2 cell were obtained. LIVE/DEAD fluorescence staining was used to confirm the viability of internalized GAS. ResultsLSFX significantly reduced the number of cell-internalized M1 and #2 GAS strains compared to the control (p < 0.01) in a dose-dependent manner. However, AMPC did not reduce this in both strains. Both live and dead intracellular GAS were confirmed in HEp-2 cells exposed to LSFX. In contrast, intracellular GAS survived in HEp-2 cells exposed to AMPC and in the control. ConclusionLSFX elicits significant bactericidal effects on cell-internalized GAS, hence it may represent a potent therapeutic option for patients with acute pharyngotonsillitis in whom AMPC treatment has failed.

Arctigenin induces caspase-dependent apoptosis in FaDu human pharyngeal carcinoma cells.

The present study was carried out to investigate the effect of Arctigenin on cell growth and the mechanism of cell death elicited by Arctigenin were examined in FaDu human pharyngeal carcinoma cells. To determine the apoptotic activity of Arctigenin in FaDu human pharyngeal carcinoma cells, cell viability assay, DAPI staining, caspase activation analysis, and immunoblotting were performed. Arctigenin inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Arctigenin-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was up-regulated by Arctigenin treatment. Moreover, caspase-8, a part of the extrinsic apoptotic pathway, was activated by Arctigenin treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria-dependent intrinsic apoptosis pathway, significantly decreased following Arctigenin treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9, and tumor suppressor -53 increased by Arctigenin treatments. In addition, Arctigenin activated caspase-3 and poly (ADP-ribose) polymerase (PARP) induced cell death. Arctigenin also inhibited the proliferation of FaDu cells by the suppression of p38, NF-κB, and Akt signaling pathways. These results suggest that Arctigenin may inhibit cell proliferation and induce apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway.

Polyurethane Structures Used as a Drug Carrier for Epigallocatechin Gallate

The current study was proposed in order to obtain, to characterize by usually physico-chemical methods and to assess in vitro, the potential cytotoxic effect of polyurethane microstructures loaded with epigallocatechin gallate (PU_MS) on human pharyngeal carcinoma cells (Detroit 562) and squamous cell carcinoma (SCC-4). The results showed that polyurethane microstructures obtained are stable and have dimensions that make them biocompatible with the biological environment. The cytotoxicity of test samples is dependent on concentration and 72 hours after stimulation at the highest concentration tested the viability of tumor cells was below 50%, the decrease being much more pronounced in the case of squamous cell carcinoma. The novel nanostructures loaded with epigallocatechin gallate induced an augmented cytotoxic effect, suggesting that this drug carriers are suitable to enhance the antitumor effect of epigallocatechin gallate.

Inhibition of cell growth and induction of apoptosis by acacetin in FaDu human pharyngeal carcinoma cells

Acacetin, which is present in damiana (Turnera diffusa ) and black locust (Robinia pseudoacacia ), has several pharmacologic activities such as antioxidant, anti-inflammatory, and anti-proliferative effects on cancer cells. However, the effect of acacetin on head and neck cancers has not been clearly established. This study aimed to examine the effects of acacetin on cell growth and apoptosis induction in FaDu human pharyngeal carcinoma cells. These were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, Live/Dead cell assay, 4′,6-diamidino-2-phenylindole dihydrochloride staining, caspase-3 and caspase-7 activation assay, and immunoblotting in FaDu cells. Acacetin induced FaDu cell death in a dose-dependent manner, with an estimated IC50 value of 41.9 µM, without affecting the viability of L-929 mouse fibroblasts as normal cells. Acacetin treatment resulted in nuclear condensation in the FaDu cells. It promoted the proteolytic cleavage of procaspase-3, -7, -8, and -9 with increasing amounts of the cleaved caspase isoforms in FaDu cells. Acacetin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting showed downregulation of the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL, but upregulation of the mitochondria-dependent pro-apoptotic proteins Bax and Badin FaDu cells after acacetin treatment. These findings indicate that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway.

Photocytotoxicity of liposomal zinc phthalocyanine in oral squamous cell carcinoma and pharyngeal carcinoma cells.

Aim: Photodynamic therapy utilizes a light-sensitive molecule that produces reactive oxygen species following irradiation. Photodynamic activities of free Zn phthalocyanine (ZnPc) and its liposomal formulations on human oral squamous cell carcinoma and pharyngeal carcinoma cells were assessed. Materials & methods: ZnPc was incorporated in extruded and nonextruded liposomes composed of palmitoyloleoylphosphatidylglycerol (POPG):palmitoyloleoylphosphatidylcholine (POPC) or POPG:dioleoylphosphatidylethanolamine liposomes and incubated with CAL 27 or FaDu cells. Cell viability was assessed following illumination and further incubation. Results: ZnPc incorporated in extruded POPG:POPC liposomes caused extensive cytotoxicity, while ZnPc in extruded or nonextruded POPG:dioleoylphosphatidylethanolamine liposomes or in multilamellar POPG:POPC liposomes were not effective. Conclusion: Extruded POPG:POPC liposomes are a useful delivery vehicle for ZnPc in photodynamic therapy of oral and pharyngeal cancers.

Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) have a high mortality rate, although several potential therapeutic targets have already been identified. Gonadotropin-releasing hormone receptor (GnRH-R) expression is less studied in head and neck cancers, hence, we investigated the therapeutic relevance of GnRH-R targeting in HNSCC patients. Our results indicate that half of the patient-derived samples showed high GnRH-R expression, which was associated with worse prognosis, making this receptor a promising target for GnRH-based drug delivery. Photodynamic therapy is a clinically approved treatment for HNSCC, and the efficacy and selectivity may be enhanced by the covalent conjugation of the photosensitizer to a GnRH-R targeting peptide. Several native ligands, gonadotropin-releasing hormone (GnRH) isoforms, are known to target GnRH-R effectively. Therefore, different 4Lys(Bu) modified GnRH analogs were designed and conjugated to protoporphyrin IX. The receptor binding potency of the novel conjugates was measured on human pituitary and human prostate cancer cells, indicating only slightly lower GnRH-R affinity than the peptides. The in vitro cell viability inhibition was tested on Detroit-562 human pharyngeal carcinoma cells that express GnRH-R in high levels, and the results showed that all conjugates were more effective than the free protoporphyrin IX.

Role of p38-mitogen-activated protein kinase in modulation of the response to therapy in FaDu Human pharyngeal carcinoma cell

Role of p38-mitogen-activated protein kinase in modulation of the response to therapy in FaDu Human pharyngeal carcinoma cell

Highly Biocompatible, Fluorescence, and Zwitterionic Carbon Dots as a Novel Approach for Bioimaging Applications in Cancerous Cells.

Highly biocompatible, excellently photostable, nitrogen- and sulfur-containing novel zwitterionic carbon dots (CDs) were synthesized by microwave-assisted pyrolysis. The size of CDs were 2-5 nm, with an average size of 2.61 ± 0.7 nm. CDs were characterized by UV/vis spectroscopy, fluorescence spectroscopy, zeta potential, Fourier-transform infrared spectroscopy, X-ray diffraction, and time-resolved fluorescence spectroscopy. CDs were known to emit blue fluorescence when excited at 360 nm, that is, UV region, and emit in the blue region of visible spectrum, that is, at 443 nm. CDs showed excitation-independent photoluminescence behavior and were highly fluorescent even at lower concentration under UV light. These CDs were highly fluorescent in nature, with the quantum yield being as high as 80%, which is comparable to that of organic dyes. The CDs were further used to image two different oral cancer cell lines, namely, FaDu (human pharyngeal carcinoma) and Cal-27 (human tongue carcinoma). The cell viability assay demonstartedthat CDs were highly biocompatible, which was further confirmed by the side scattering studies as no change in the granularity was observed even at the highest concentration of 1600 μg/mL. The generation of reactive oxygen species (ROS) was also investigated and negligible generatonof ROS was detected. In addition to that, the uptake phenomenon, cell cycle analysis, exocytosis, and cellular uptake at 4 °C and in the presence of ATP inhibitor were studied. It was found that CDs easily cross the plasma membrane without hampering the cellular integrity.

Abstract LB-149: Discovery and characterization of fully human monoclonal antibodies that neutralize multiple TGFβ isoforms

Abstract TGFβ levels are elevated in, and can drive the progression of, numerous disease states including advanced metastatic cancer and fibrosis. The exact role of each TGFβ isoform in disease is an area of active research. To help elucidate the therapeutic importance and test the value of inhibiting multiple TGFβ isoforms, we generated antibodies with differing specificities to each of the three TGFβ isoforms. Antibodies that functionally neutralized TGFβ1/2, TGFβ2/3, and TGFβ1/2/3 were characterized in vitro, and in vivo. Two surface plasmon resonance (SPR)-based kinetic methods were used to compare the binding affinities and rate constants of the three antibodies. All three antibodies had single digit nanomolar affinities (KD) or better to the TGFβ isoforms, ranging from 1.7pM to 1400pM. The antibodies were characterized in cell based assays for their ability to effect signaling, cell proliferation, and stimulated cytokine release, and EC50 values for each were obtained. Potency in each of the cell assays directly correlated with the affinity of the antibody for the TGFβ isoform used to elicit the effect.These antibodies were then used to elucidate the role of each of the TGFβ isoforms in more complex systems in vivo. Detroit 562, a human metastatic pharyngeal carcinoma derived cell line, was shown to express all three TGFβ isoforms in vitro and in vivo. 5x106 cells were implanted subcutaneously on the flanks of athymic (nu/nu) mice. Tumors were established to ∼200 mm3, and treatment initiated at day 7 post implant. Groups were treated with 10mg/kg IP twice weekly. At Day 28, blocking all three TGFβ isoforms with a pan reactive TGFβ1/2/3 antibody significantly reduced mean tumor volume (219 ± 38 mm3) compared to a control IgG (1022 ± 241mm3) (n= 12 mice per group). Additionally, dose-ranging studies were performed (1, 3, or 10 mg/kg) to determine the relative potency of the antibodies. We found that tumors in mice treated with either the anti-TGFβ1/2 or the pan anti-TGFβ1/2/3 inhibitor antibody (3mg/kg twice weekly) showed notable and significant (p&amp;lt;0.005) differences in mean tumor volumes and mean tumor weights at day 30 compared to IgG control antibody, with average tumor volume reductions of 63%, and 64% respectively. In this model, inhibition of tumor growth by the anti-TGFβ1/2 antibody was indistinguishable from inhibition by the anti-TGFβ1/2/3 antibody, suggesting that TGFβ3 may not play a role in tumor progression. Conversely, in this model, inhibition of TGFβ2/3 alone was insufficient to significantly impact tumor growth. These data suggest that effective neutralization of TGFβ1/2 function may be sufficient to inhibit tumor growth and that neutralizing all three TGFβ isoforms is not required. Specific and targeted inhibition of select TGFβ isoforms offers a more focused therapeutic approach which has potential for improved safety. Experiments are underway to elucidate the role of the various TGFβ isoforms in key aspects of tumor biology. Citation Format: Amer Mirza, Daniel Bedinger, Llewelyn Lao, Shireen Khan, Toshihiko Takeuchi. Discovery and characterization of fully human monoclonal antibodies that neutralize multiple TGFβ isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-149. doi:10.1158/1538-7445.AM2014-LB-149

Antitumor Activity from the Combined Application of Poly(I:C) and Chemotherapeutics in Human Metastatic Pharyngeal Cell Lines

Background: Toll-like receptor 3 (TLR3) activation in tumor cells induces apoptosis. We investigated the effect of TLR3 ligand (poly(I:C)) in combination with chemotherapeutics applied to human pharyngeal carcinoma cells as a possible antitumor therapy. Methods:Human pharyngeal cancer cell lines were studied (FaDu and Detroit 562). Cytotoxicity assays and apoptosis assays (annexin V staining and caspase 3/7 activity measurements) were used to investigate the cytotoxic effects. By using TLR3 siRNA we confirmed that the observed effect is TLR3-dependent. Results: We found that the combined application of poly(I:C) and chemotherapeutics (cisPt, HU, 5-FU and MTX) has a stronger inhibitory effect on cell growth in tumor cells expressing functional TLR3 as compared with a single treatment. This is a result of TLR3-dependent apoptosis. Conclusion: Our study showed that a combined application of the two agents already being used in tumor therapy could lower the necessary dosage of chemotherapeutics, leading to fewer side effects.

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.

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.