CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.

BACKGROUND: Cancer stem cells (CSCs) are responsible for treatment failure. However, their identification and roles in resistance are not well established in head and neck squamous cell carcinoma (HNSCC). METHODS: Three HNSCC cell lines (FaDu, Detroit562 and BICR6) were treated with cisplatin or radiation. Cell surface antigens were analysed by LyoPlate, a novel cell surface antigen array. The expression levels of antigens highly expressed after treatments were further compared between cisplatin-resistant Detroit562 cells and its parental line. Association of the candidate antigen with CSCs properties, namely sphere formation and in vivo tumourigenicity, was also examined. RESULTS: CD10, CD15s, CD146 and CD282 were upregulated across the treated cell lines, while the increased expression of CD10 was prominent in the cisplatin-resistant cell line. Isolation mediated by FACS revealed that the CD10-positive subpopulation was more refractory to cisplatin, fluorouracil and radiation than the CD10-negative subpopulation. It also showed an increased ability to form spheres in vitro and tumours in vivo. Moreover, the CD10-positive subpopulation expressed the CSC marker OCT3/4 at a higher level than that in the CD10-negative subpopulation. CONCLUSIONS: CD10 is associated with therapeutic resistance and CSC-like properties of HNSCC. CD10 may serve as a target molecule in the treatment of refractory HNSCC.

Effectiveness of the computed analysis of electroglottographic signals in muscle tension dysphonia.

OBJECTIVE: To investigate the usefulness of electroglottography (EGG) parameters in the diagnosis and estimation of efficacy of voice therapy for muscle tension dysphonia (MTD). PATIENTS AND METHODS: Nineteen MTD participants, an equivalent number of dysphonic ('organic') patients with vocal fold lesions and as many normal speakers were enrolled. Acoustic (Ac) and EGG signals during sustained phonation were recorded simultaneously. The period and amplitude perturbation quotient of both signals, the closed quotient (CQ) of EGG signals (mean CQ) and its standard deviation (CQSD) were calculated, and subsequently compared among the three groups. These parameters in the MTD group were compared before and after voice therapy. RESULTS: The perturbation measures of both signals in the MTD group were either as high as or significantly higher than those in the organic group or the control group, respectively. Both the Ac and EGG parameters after therapy significantly decreased. The CQSD, but not mean CQ, also decreased after therapy. CONCLUSION: EGG parameters related to the regularity of vocal fold vibration, but not to the degree of vocal fold contact (mean CQ), are useful for the diagnosis and estimation of voice therapy outcome in MTD.

Prevalence of human papillomavirus in oral gargles and tonsillar washings.

OBJECTIVES: Human papillomavirus (HPV) infection drives carcinogenesis in the oropharynx. No standard sampling or HPV detection methods for evaluating oropharyngeal HPV infection exist. The prevalence of oral HPV infection in Japan is unknown. MATERIALS AND METHODS: We examined 435 healthy Japanese individuals to address whether adding tonsillar washing to oral gargling would improve HPV detection. We compared HPV assessment using GENOSEARCH HPV31 versus nested PCR and direct sequencing. Associations between HPV infection and demographic and behavioral characteristics were examined. RESULTS: Most participants who were HPV-positive based on oral gargles were also HPV-positive based on tonsillar washings: 11 (64.7%) of 17 on nested PCR and 12 (70.6%) of 17 on GENOSEARCH HPV31. Although HPV infection was more prevalent in oral gargles followed by tonsillar washings than in oral gargles alone, the difference was not statistically significant (nested PCR, 4.8% vs. 3.9%, P = 0.46; GENOSEARCH HPV31, 5.3% vs. 3.9%, P = 0.33). The overall agreement between nested PCR and GENOSEARCH HPV31 was 98.6%, with 76.0% positive agreement. The overall prevalence of oral HPV infection in Japan was 5.7% (95% confidence interval, 3.9-8.3%). Men had a significantly higher prevalence of oral HPV infection than women (8.3% vs. 2.6%, P = 0.02). Infection increased with number of lifetime sexual partners (P < 0.001 for trend). CONCLUSION: The oropharynx is probably the major source of HPV-infected cells in oral gargles. Oral gargling could be a standard sampling method for evaluating oropharyngeal HPV infection. GENOSEARCH HPV31 could be an option for oral HPV detection.

Metabolic tumor volume of metastatic lymph nodes and survival after total laryngectomy in laryngeal and hypopharyngeal cancer.

OBJECTIVES: The prognostic value of metabolic tumor volume (MTV) in locally advanced laryngeal or hypopharyngeal cancer is established in the setting of chemoradiotherapy, while it remains unknown in the setting of upfront total laryngectomy. MATERIALS AND METHODS: We retrospectively analyzed 88 patients receiving total laryngectomy and neck dissection, using Cox regression models. RESULTS AND CONCLUSION: Variables related to metastatic lymph node were associated with overall survival, whereas those related to primary tumor were not. In multivariable models, MTV of metastatic lymph nodes (N-MTV) as a continuous variable (Akaike's information criterion (AIC), 277.5) was equivalent to pathological nodal status (AIC, 278.2; P = 0.40), and superior to pathological nodal classification as an ordinal variable (AIC, 281.4; P < 0.05) in ability of predicting death. The risk of death was increased by 1.2-fold (95% confidence interval (CI), 1.0-1.4; P = 0.03) every 10-ml increment of N-MTV, while patients with pN+ disease were at a higher risk of death by 2.9-fold (95% CI, 1.0-12.2; P < 0.05) compared with patients with pN0 disease. Using recursive partitioning analysis (RPA), we classified the patients as having a low, intermediate, or high risk of death on the basis of N-MTV and extranodal extension (ENE). This RPA classification system exhibited greater concordance with overall survival than the classification considering pathological nodal status and ENE (AIC, 275.8 versus 281.4; P = 0.02). In the setting of upfront total laryngectomy, N-MTV is a critical predictor of mortality. A staging system in which N-MTV is incorporated may better inform adjuvant treatment decisions.

Functional evidence that cell surface galectin-3 mediates homotypic cell adhesion.

Galectin-3 (Gal-3) is a beta-galactoside-binding protein with M(r) approximately 30,000. Cell surface Gal-3 is postulated to be involved in homotypic aggregation of tumor cells in the circulation during metastasis through attachment to a complementary serum glycoprotein(s), which serves as a cross-linking bridge between adjacent cells. To test this hypothesis a recombinant strain of baculovirus encoding Gal-3 was used to infect Sf9 insect cells, which lack endogenous Gal-3. Immunoblotting and indirect immunofluorescence studies revealed that the infection with recombinant virus conferred Gal-3 expression on Sf9 cells, and the Gal-3 was localized on the cell surface as well as in the cytoplasm. Sf9 cells infected with recombinant virus underwent homotypic aggregation in the presence of exogenous glycoprotein (i.e., asialofetuin), whereas control cells uninfected or infected with wild-type virus did not. Lactose and Fab' fragments of anti-Gal-3 antibodies markedly inhibited the cell-cell aggregation. Moreover, cosuspension of Sf9 cells infected with the recombinant virus with uninfected cells in the presence of asialofetuin resulted in a preferential cell-cell adhesion of the Gal-3-expressing cells. These results directly demonstrate the ability of cell surface Gal-3 molecules to mediate homotypic cell adhesion by bridging through branched, soluble complementary glycoconjugates.

Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion.

Galectin-3 is a beta-galactoside-specific lectin implicated in diverse processes involved in cellular interactions. Recently, the Mac-2-binding protein, a heavily N-glycosylated secreted protein with a subunit Mr of 97,000, was identified as its ligand. The present study characterizes the interaction between galectin-3 and Mac-2-binding protein in whole cells and measures their relative expression levels. Incubation of A375 cells with affinity-purified Mac-2-binding protein resulted in its binding to galectin-3 on the cell surface in a specific carbohydrate-dependent manner. Mac-2-binding protein also induced homotypic cell aggregation, which was inhibited by lactose or Fab' fragments of an anti-galectin-3 antibody. Northern blotting analysis revealed differences in the transcriptional regulation of galectin-3 and Mac-2-binding protein. These results provide the first direct evidence for a Mac-2-binding protein function and suggest that it may play a role in tumor cell embolization during metastasis through interaction with galectin-3.

Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family.

Galectin-3, a beta-galactoside-binding protein, has been shown to be involved in tumor progression and metastasis. Here, we demonstrate that expression of galectin-3 in human breast carcinoma BT549 cells inhibits cis-diamminedichloroplatinum (cisplatin)-induced poly(ADP-ribose) polymerase degradation and apoptosis, without altering Bcl-2, Bcl-X(L), or Bax expressions. Galectin-3 contains the NWGR amino acid sequence highly conserved in the BH1 domain of the bcl-2 gene family, and a substitution of glycine to alanine in this motif abrogated its antiapoptotic activity. Our findings demonstrate that galectin-3 inhibits apoptosis through a cysteine protease pathway and highlight the functional significance of the NWGR motif in apoptosis resistance of a non-Bcl-2 protein.

Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells.

Galectin-3 is an endogenous beta-galactoside-binding protein with specificity for type I and II ABH blood group epitopes and poly-N-acetyllactosamine glycan-containing cell surface glycoproteins and is the major nonintegrin cellular laminin-binding protein. Galectin-3 is expressed at an elevated level in a wide range of neoplasms, and expression was shown to be associated in some tumor cell systems with metastases. Here we determined the functional consequence of blocking galectin-3 expression in highly malignant human breast carcinoma MDA-MB-435 cells. Inhibition of galectin-3 expression led to reversion of the transformed phenotype as determined by altered morphology, loss of serum-independent growth, acquisition of growth inhibition properties by cell contact, and abrogation of anchorage-independent growth. The blockage of galectin-3 expression led to a significant suppression of tumor growth in nude mice. These results provide direct evidence that galectin-3 expression is necessary for the maintenance of the transformed and tumorigenic phenotype of MDA-MB-435 breast carcinoma cells.

Expression of cytoplasmic galectin-3 as a prognostic marker in tongue carcinoma.

Galectin-3 is a member of the beta-galactoside-binding mammalian lectin family with affinity to ABH group epitopes, cell surface and extracellular polylactosamine glycans. It has been shown to be involved in differentiation, morphogenesis, tumor progression, and metastasis. Here we questioned the possible involvement of galectin-3 in the neoplastic progression of the tongue epithelium and evaluated its prognostic value in tongue cancer patients. Galectin-3 expression was analyzed by the immunohistochemical method in 77 tongue specimens (54 squamous cell carcinomas and 23 specimens of distinct normal mucosa). Levels of nuclear expression of galectin-3 markedly decreased during the progression from normal to cancerous states (P < 0.0001), while cytoplasmic expression increased (P < 0.0001). Enhanced expression of galectin-3 in the cytoplasm was associated with a reduced disease-free survival of tongue cancer patients. Multivariate analysis identified enhanced expression of cytoplasmic galectin-3 as an independent predictor of disease recurrence (P = 0.0120). These results suggest that the observed translocation of galectin-3 from the nucleus to the cytoplasm during neoplastic progression may serve as a prognostic factor for tongue cancer patients.

Galectin-3 maintains the transformed phenotype of thyroid papillary carcinoma cells.

Galectin-3, a beta-galactoside-binding protein, is highly expressed in thyroid papillary carcinomas, while functional relevance of galectin-3 overexpression to the malignant phenotype remains elusive. In the present study we transfected galectin-3 antisense cDNA into the human thyroid papillary carcinoma cell line NPA which expresses an innately high level of galectin-3, and examined the effect of antisense inhibition of galectin-3 expression on the transformed phenotype. There was no difference in anchorage-dependent growth between the antisense clones and either the control or parental clones. In contrast, anchorage-independent growth and saturation density of the antisense clones were significantly suppressed compared to those of either the control or parental clones. These results demonstrate that overexpression of galectin-3 in thyroid papillary carcinoma cells is necessary for the maintenance of transformed phenotype, and suggest galectin-3 as a potential target for therapeutic interventions in the future.

SCID (severe combined immunodeficiency) mice as a new system to investigate metastasis of human tumors.

In severe combined immunodeficiency (scid) mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) grew rapidly to enormous sizes in all of the animals after both subcutaneous and intraperitoneal transplantation, while only half of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice. Furthermore, transplanted tumors metastasized spontaneously to distant organs such as lung, liver, kidney, pancreas, and spleen in scid mice, while metastases were not found in athymic nude mice. Similar results were observed in scid mice and scid-nude (streaker) double mutant mice with human classic (typical) seminoma which has been neither transplantable nor metastatic in athymic nude mice. Thus, scid mice provide an invaluable experimental system to investigate the mechanism of metastasis which is the most important and life-threatening problem in cancer patients.

Association of p53 expression with second primary tumour development in hypopharyngeal carcinoma.

Abnormalities of p53 tumour suppressor gene are detected in a diversity of malignancies and play an important role in their pathogenesis. Hypopharyngeal carcinoma is the most morbid among head and neck squamous cell carcinomas because of the high incidence of treatment failures and because a biological marker predictive of the treatment failures remains elusive. The expression of p53 protein in 46 hypopharyngeal squamous cell carcinomas was examined histochemically and p53 immunoreactivity was found in 19 of 46 cases (41.3 per cent). The rate of second primary tumour development was significantly higher in the p53-positive group than in the p53-negative group (p = 0.039), whereas that of tumour recurrence was not significantly different between the two. Moreover, there was no statistically significant difference in either overall or disease-free survival between the p53-positive and -negative groups. These results indicate that although p53 expression significantly correlates with second primary tumour development in patients with hypopharyngeal squamous cell carcinomas, it is not predictive of the clinical outcome.

Expression and translocation of aquaporin-2 in the endolymphatic sac in patients with Meniere's disease.

Meniere's disease, characterised by episodic vertigo, fluctuating hearing loss and tinnitus, can occur under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although its pathogenesis has been proposed to be a disorder of water transport in the inner ear, subsequently, it remains unsolved, until now. A recent study revealed that both plasma stress hormone, vasopressin (pAVP) and its receptor, V2 (V2R) expression in the inner ear endolymphatic sac were significantly higher in Meniere's patients. In the present study, to link V2R-related molecules and inner ear hydrops, we examined V2R-linked water channel molecule, aquaporin-2 (AQP2) expression and translocation in human endolymphatic sac. AQP2 mRNA expression in the endolymphatic sac was significantly higher in Meniere's patients by using real-time polymerase chain reaction, as further confirmed by western blotting. AQP2-like immunoreactivity (-LIR) was translocated from luminal to basolateral side with endosomal trapping in the endolymphatic sac at the time of AVP exposure in human endolymphatic sac tissue culture. The similar AQP2-LIR translocation was also demonstrated by forskolin and blocked by vasopressin/V2R specific antagonist, OPC31260 and protein kinase A (PKA) specific antagonists, H-89 and KT-5720. We concluded that in the pathogenesis of inner ear hydrops resulting in Meniere's attacks, pAVP elevation as a result of stress and subsequent V2R-cAMP-PKA-AQP2 activation and endosomal trapping of AQP2 in the endolymphatic sac, might be important as a basis of this disease. Further experimental and clinical studies are needed to better clarify the neuroscientific relationship between stress and Meniere's disease.

Behavioral assessment and identification of a molecular marker in a salicylate-induced tinnitus in rats.

Tinnitus is a non-observable phantom sensation. As such, it is a difficult condition to investigate and, to date, no effective treatment has been developed. To approach this phantom sensation, we aimed to develop a rat behavioral model of tinnitus using salicylate, an active component of aspirin known to induce tinnitus. We also aimed to establish a molecular marker of tinnitus by assessing the expression of transient receptor potential cation channel superfamily V-1 (TRPV1) in the rat auditory pathway during salicylate-induced tinnitus. Animals were trained to perform "an active avoidance task": animals were conditioned by electrical footshock to move to the other side of the conditioning box when hearing a sound. Animals received a single injection of saline or salicylate (400 mg/kg i.p.) and false positive responses were measured 2 h after injection as the number of movements during a silent period. The number of responses in salicylate-treated animals was highest when the conditioned stimulus was 60 dB sound pressure level (SPL) and 16 kHz. This indicates that animals could feel tinnitus 2 h after salicylate injection, equivalent to that induced by 60 dB SPL and 16 kHz. By means of real-time PCR and western blot analysis, TRPV1 expression was significantly upregulated in spiral ganglion cells 2 h after salicylate injection and this upregulation together with the increase in the number of false positive responses was significantly suppressed by capsazepine (10 mg/kg i.p.), a specific antagonist of TRPV1. This suggests that salicylate could induce tinnitus through activation of TRPV1 in the rat auditory pathway.

A rare case of a glass foreign body in the parapharyngeal space: pre-operative assessment by contrast-enhanced CT and three-dimensional CT images.

The parapharyngeal space is an infrequent area for foreign bodies to lodge. However, the presence of trauma or inflammation near or within the space is dangerous because of its anatomical proximity to the bifurcation of the maxillary artery, carotid artery and jugular vein. We encountered a rare case, when a glass flask burst, in which intraparotid damage to the facial nerve was seen, as well as a glass foreign body lodged in the parapharyngeal space close to the above named great vessels. We emphasise the usefulness of contrast-enhanced CT and three-dimensional CT images for pre-operative evaluation of the locational relationship between the foreign body and great vessels in the parapharyngeal space.

Effects of natural complex carbohydrate (citrus pectin) on murine melanoma cell properties related to galectin-3 functions.

Citrus pectin (CP) and pH-modified citrus pectin (MCP) are highly branched and non-branched complex polysaccharides, respectively, rich in galactoside residues, capable of combining with the carbohydrate-binding domain of galectin-3. We reported previously that intravenous injection of B16-F1 murine melanoma cells with CP or MCP into syngeneic mice resulted in a significant increase or decrease of lung colonization, respectively (Platt D, Raz A (1992) J Natl Cancer Inst 84:438-42). Here we studied the effects of these polysaccharides on cell-cell and cell-matrix interactions mediated by carbohydrate-recognition. MCP, but not CP, inhibited B16-F1 melanoma cells adhesion to laminin and asialofetuin-induced homotypic aggregation. Both polysaccharides inhibited anchorage-independent growth of B16-F1 cells in semisolid medium, i.e. agarose. These results indicate that carbohydrate-recognition by cell surface galectin-3 may be involved in cell-extracellular matrix interaction and play a role in anchorage-independent growth as well as the in vivo embolization of tumour cells.

Identification of human melanoma cellular and secreted ligands for galectin-3.

The endogenous human tumor-associated galectin-3 (hL-31) is a functional molecule which acts as a receptor for ligands containing poly-N-acetyllactosamine sequences. However, little is known about its native ligand(s). In order to identify the ligand(s), the human melanoma cell line A375 was metabolically labeled with [3H]glucosamine, and total cell extracts and serum-free conditioned medium of the labeled cells were affinity-purified on immobilized recombinant hL-31 followed by elution with lactose, the specific sugar inhibitor of the lectin. Cellular ligands for hL-31 were found to be composed of the two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, while secreted ligands consisted of two glycoproteins of 98 and 70 kDa. N-terminal protein microsequencing revealed that the 98 kDa and 70 kDa species share the same N-terminal sequence. The functional relevance of these secreted ligands was demonstrated by their ability to inhibit lectin-mediated hemagglutination in a manner similar to the specific sugar inhibitor lactose. Computer-assisted sequence library searches have identified the 98 kDa human melanoma secreted ligand to be the Mac-2-binding protein (Mac-2-BP), also known as the human lung tumor L3 antigen.