Development of an in vitro cell-sheet cancer model for chemotherapeutic screening.

Epithelial cancer grows in vivo in a microenvironment that comprises tumour, stroma, and immune cells. A three-dimensional (3D) culture model might be able to mimic the tumour microenvironment in vivo; therefore, we developed a new 3D epithelial cancer model using in vitro cell-sheet engineering and compared the results of treatment with several chemotherapeutic drugs among the 3D cell-sheet model, spheroid culture, and 2D cell culture. Methods: The cell sheet comprised keratinocytes and a plasma fibrin matrix containing fibroblasts. Cancer spheroids with or without cancer-associated fibroblasts (CAFs) were interposed between the keratinocytes and fibrin layer. Cell growth, viability, and hypoxia were measured using the cell counting kit-8, LIVE/DEAD assay, and propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expression were compared among the different cell culture models. Results: Enhanced resistance to sorafenib and cisplatin by cancer spheroids and CAFs was more easily observed in the 3D than in the 2D model. Invasion by cancer-CAF spheroids into the fibrin matrix was more clearly observed in the 3D cell sheet. The expansion of viable cancer cells increased in the 3D cell sheet, particularly in those with CAFs, which were significantly inhibited by treatment with 10 µM sorafenib or 20 µM cisplatin (P < 0.05). TGF-ß1, N-cadherin, and vimentin mRNA and protein levels were higher in the 3D cell-sheet model. Conclusions: The 3D cell sheet-based cancer model could be applied to in vitro observation of epithelial cancer growth and invasion and to anticancer drug testing.

Hederagenin Induces Apoptosis in Cisplatin-Resistant Head and Neck Cancer Cells by Inhibiting the Nrf2-ARE Antioxidant Pathway.

Acquired resistance to cisplatin is the most common reason for the failure of cisplatin chemotherapy. Hederagenin, triterpenoids extracted from ivy leaves, exhibits antitumor activity in various types of cancer. However, the therapeutic potential of hederagenin in head and neck cancer (HNC) has remained unclear. Therefore, we examined the effects of hederagenin in cisplatin-resistant HNC cells and characterized its molecular mechanisms of action in this context. We evaluated the effects of hederagenin treatment on cell viability, apoptosis, reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential (ΔΨm), and protein and mRNA expression in HNC cells. The antitumor effect of hederagenin in mouse tumor xenograft models was also analyzed. Hederagenin selectively induced cell death in both cisplatin-sensitive and cisplatin-resistant HNC cells by promoting changes in ΔΨm and inducing apoptosis. Hederagenin inhibited the Nrf2-antioxidant response element (ARE) pathway and activated p53 in HNC cells, thereby enhancing ROS production and promoting glutathione depletion. These effects were reversed by the antioxidant trolox. Hederagenin activated intrinsic apoptotic pathways via cleaved PARP, cleaved caspase-3, and Bax. The selective inhibitory effects of hederagenin were confirmed in cisplatin-resistant HNC xenograft models. These data suggest that hederagenin induces cell death in resistant HNC cells via the Nrf2-ARE antioxidant pathway.

Targeting Nrf2 with wogonin overcomes cisplatin resistance in head and neck cancer.

A principal limitation to the clinical use of cisplatin is the high incidence of chemoresistance to this drug. Combination treatments with other drugs may help to circumvent this problem. Wogonin, one of the major natural flavonoids, is known to reverse multidrug resistance in several types of cancers. We investigated the ability of wogonin to overcome cisplatin resistance in head and neck cancer (HNC) cells and further clarified its molecular mechanisms of action. Two cisplatin-resistant HNC cell lines (AMC-HN4R and -HN9R) and their parental and other human HNC cell lines were used. The effects of wogonin, either alone or in combination with cisplatin, were assessed in HNC cells and normal cells using cell cycle and death assays and by measuring cell viability, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Wogonin selectively killed HNC cells but spared normal cells. It inhibited nuclear factor erythroid 2-related factor 2 and glutathione S-transferase P in cisplatin-resistant HNC cells, resulting in increased ROS accumulation in HNC cells, an effect that could be blocked by the antioxidant N-acetyl-L-cysteine. Wogonin also induced selective cell death by targeting the antioxidant defense mechanisms enhanced in the resistant HNC cells and activating cell death pathways involving PUMA and PARP. Hence, wogonin significantly sensitized resistant HNC cells to cisplatin both in vitro and in vivo. Wogonin is a promising anticancer candidate that induces ROS accumulation and selective cytotoxicity in HNC cells and can help to overcome cisplatin-resistance in this cancer.

Long-term voice outcomes after thyroplasty for unilateral vocal fold paralysis.

OBJECTIVES: To investigate the long-term clinical efficacy and stability of thyroplasty type I for unilateral vocal cord palsy, and to identify the appropriate timing of posttreatment evaluations for determination of long-term voice outcome. Study DESIGN: Single-institution retrospective study. SETTING: Academic tertiary referral centers in Korea. PATIENTS: Forty patients with unilateral vocal cord palsy who underwent thyroplasty from January 1, 1996, through December 31, 2006, and were followed up for at least 5 years after the surgical procedure. INTERVENTIONS: Thyroplasty type I under local anesthesia. MAIN OUTCOME MEASURES: Acoustic and aerodynamic analyses of voice were performed on the day before the operation and at preset intervals afterward. Two blinded speech-language pathologists performed the perceptual evaluation. RESULTS: The GRBAS scale (grade of hoarseness, roughness, breathiness, asthenia, and strain) values showed significant improvement at 6 months after the operation (P < .05); these improvements continued up to 1 year and were maintained 5 years after the operation. Acoustic measurements of shimmer and jitter began to show significant improvement at 6 months after the operation, and fundamental frequency and noise harmonic ratios evidenced significant improvement at 1 year (P < .05); these improvements were maintained, to a significant extent, at 5 years after the operation. Aerodynamically, the maximum phonation time, glottal flow rate, and peak subglottic pressure improved significantly from before the operation to 6 months and 1 year after the operation, attaining near-normal values at 1 year afterward (P < . 05) CONCLUSIONS: Thyroplasty type I may provide evidence that voice outcome progressively evolves during the first years after the surgical procedure, and that subsequent vocal improvement presented long-lasting stabilization. To assess the long-term voice quality, it may be enough to perform the voice evaluation at 1 year after the procedure.

Zinc deficiency may be a cause of burning mouth syndrome as zinc replacement therapy has therapeutic effects.

BACKGROUND: Zinc is known to play an important role for growth and development, the immune response, neurological function, and reproduction. Although the etiology of burning mouth syndrome (BMS) is unknown, zinc deficiency may be implicated in the pathogenesis of BMS. The aim of this study was to demonstrate a causal relationship between zinc deficiency and BMS and to assess whether zinc replacement is an effective therapy for BMS. METHODS: Serum zinc level was evaluated in 276 patients with BMS. To assess the therapeutic effect of zinc replacement, patients with zinc deficiency were administered a zinc supplement (14.1 mg/day). Pain intensity 6 months after zinc replacement was evaluated using an 11-point numerical scale. We also developed an animal model of zinc deficiency to assess the effects of zinc deficiency on the oral mucosa. RESULTS: Of the 276 patients with BMS, 74 (26.8%) had low serum zinc levels. Zinc replacement therapy lowered the mean numerical pain scale in these patients from 8.1 to 4.1, compared with a mean decrease from 7.7 to 6.7 in a control group (P = 0.004). In our animal model of zinc deficiency, the main pathologic findings were hyperkeratinization and increased mitosis on the dorsum of the tongue, although there were no gross oral mucosal lesions. CONCLUSIONS: Zinc deficiency might play a role in some patients with BMS. In such patients, appropriate zinc replacement therapy is effective in relieving symptoms.

Prevention of postoperative hypocalcemia with routine oral calcium and vitamin D supplements in patients with differentiated papillary thyroid carcinoma undergoing total thyroidectomy plus central neck dissection.

BACKGROUND: Routine oral calcium and vitamin D supplementation may prevent hypocalcemic crisis, but its efficacy has not been studied in patients undergoing thyroidectomy plus central neck dissection (CND). The authors therefore prospectively evaluated the clinical usefulness of routine oral calcium and vitamin D supplementation for prevention of hypocalcemia after total thyroidectomy and CND. METHODS: Of 197 patients with differentiated papillary thyroid carcinoma, 49 underwent total thyroidectomy alone, and 148 underwent total thyroidectomy plus CND. The latter were randomized to oral calcium (3 g/day) plus vitamin D (1 mg/day) (Group A, n=49), calcium alone (Group B, n=49), or no supplements (Group C, n=50). Hypocalcemic symptoms, serum calcium, and parathyroid hormone (PTH) levels were compared among the groups. RESULTS: Group C had significantly higher incidences of symptomatic (26.0% vs 6.1%; P<.015) and laboratory (44.0% vs 14.3%; P<.015) hypocalcemia than the group without CND. The incidences of symptomatic and laboratory hypocalcemia were significantly decreased in Groups A (2.0% and 8.2%, respectively) and B (12.2% and 24.5%, respectively) (P<.05). Serum calcium levels decreased in most patients after surgery, but recovered earliest in Group A. Hypercalcemia and PTH inhibition did not occur in gs A and B. CONCLUSIONS: Compared with total thyroidectomy alone, CND significantly increases the rate of postoperative hypocalcemia, which can be prevented by routine postoperative supplementation with oral calcium and vitamin D.

The effect of electrical stimulation therapy on dysphagia following treatment for head and neck cancer.

The purpose of this study was to evaluate the effect of neuromuscular electrical stimulation (NMES) in patients suffering from dysphagia following treatment for head and neck cancer. In a prospective, double blinded, randomized case control study between January 2006 and December 2007, 14 patients were randomized to 30min of NMES and 30min of traditional swallowing training for 5 days per week for 2 weeks (experimental group), and 12 patients were randomized to sham stimulation plus traditional swallowing training (control group). Effects were assessed using the clinical dysphagia scale (CDS), the functional dysphagia scale (FDS), the American speech-language-hearing association national outcome measurement system (ASHA NOMS) and the M.D. Anderson dysphagia inventory (MADI). Pretreatment evaluation showed no significant differences between the two groups for all parameters. Average changes of FDS score were 11.4+/-8.1 for the experimental group and 3.3+/-14.0 for the control group (P=0.039). CDS, ASHA NOMS and MADI showed some difference with treatment, but the changes were not significant (P>0.05). NMES combined with traditional swallowing training is superior to traditional swallowing training alone in patients suffering from dysphagia following treatment for head and neck cancer.

Routine oral calcium and vitamin D supplements for prevention of hypocalcemia after total thyroidectomy.

BACKGROUND: The purpose of this study was to evaluate the clinical usefulness of routine oral calcium and vitamin D supplements in the prevention of hypocalcemia after total thyroidectomy. METHODS: Ninety patients who underwent total thyroidectomy were randomly assigned to routinely receive or not receive a supplement containing oral calcium (3 g/d) and vitamin D (1 g/d) for 2 weeks. Hypocalcemic signs and symptoms, serum calcium, and parathyroid hormone (PTH) levels were monitored and compared between the 2 groups. RESULTS: The incidences of symptomatic and laboratory hypocalcemia were significantly lower in the oral calcium/vitamin D group than in the group not receiving the supplement: 3 of 45 patients (7%) versus 11 of 45 (24%) and 6 of 45 (13%) versus 16 of 45 (36%), respectively (P < or = .02). The hypocalcemic symptoms were minimal in the supplement group but more severe in the group not receiving the supplement. Serum calcium levels decreased in both groups after surgery but recovered earlier in the supplement group. No hypercalcemia or PTH inhibition developed in the supplement group. CONCLUSION: Routine administration of a supplement containing oral calcium and vitamin D is effective in reducing the incidence and severity of hypocalcemia after total thyroidectomy.

Intraoperative parathyroid hormone assay for management of patients undergoing total thyroidectomy.

BACKGROUND: Rapid parathyroid hormone (PTH) assay has been applied to predict hypocalcemia after thyroidectomy compared with conventional close monitoring of serum calcium levels. We evaluated the reliability of intraoperative intact PTH (ioPTH) assay to predict hypocalcemia after total thyroidectomy and sought to develop an algorithm for the management of postthyroidectomy patients. METHODS: Rapid PTH assays were performed before and after thyroidectomy for 92 new patients receiving total thyroidectomy. Preoperative and postoperative serum calcium and standard PTH levels were serially obtained to 6 months after surgery RESULTS: Postoperative hypocalcemia developed in 34 of 92 patients (37%), who showed significantly lower ioPTH values compared with those of normocalcemic patients (mean 9.2 pg/mL vs 31.3 pg/mL). The ioPTH levels were significantly correlated with standard PTH levels (p < .001, r > 0.62), but not with early serum calcium levels within 8 hours after the operation. Sensitivity and specificity of ioPTH levels of <15.0 pg/mL for the prediction of postoperative hypocalcemia were 85% and 84%, respectively. A value of >15.0 pg/mL and <70% decline in ioPTH after thyroidectomy can reliably identify normocalcemic patients during thyroidectomy or patients requiring close monitoring and early calcium supplement CONCLUSIONS: Rapid ioPTH assay can reliably monitor parathyroid function after thyroidectomy and predict postoperative hypocalcemia. The proposed algorithm based on rapid PTH levels will lead to improved prediction of normocalcemic patients.