Randomized trial of supplemental beta-carotene to prevent second head and neck cancer.

Beta-carotene has established efficacy in animal models of oral carcinogenesis and has been shown to regress oral precancerous lesions in humans. The purpose of this study was to see whether these effects extended to the prevention of oral/pharyngeal/laryngeal (head and neck) cancer in humans. The subject population for this randomized, placebo-controlled, double-blinded clinical trial included 264 patients who had been curatively treated for a recent early-stage squamous cell carcinoma of the oral cavity, pharynx, or larynx. Patients were assigned randomly to receive 50 mg of beta-carotene per day or placebo and were followed for up to 90 months for the development of second primary tumors and local recurrences. After a median follow-up of 51 months, there was no difference between the two groups in the time to failure [second primary tumors plus local recurrences: relative risk (RR), 0.90; 95% confidence interval (CI), 0.56-1.45]. In site-specific analyses, supplemental beta-carotene had no significant effect on second head and neck cancer (RR, 0.69; 95% CI, 0.39-1.25) or lung cancer (RR, 1.44; 95% CI, 0.62-3.39). Total mortality was not significantly affected by this intervention (RR, 0.86; 95% CI, 0.52-1.42). Whereas none of the effects were statistically significant, the point estimates suggested a possible decrease in second head and neck cancer risk but a possible increase in lung cancer risk. These effects are consistent with the effects observed in trials using intermediate end point biological markers in humans, in which beta-carotene has established efficacy in oral precancerous lesions but has no effect or slightly worsens sputum cytology, and in animal carcinogenicity studies, in which beta-carotene has established efficacy in buccal pouch carcinogenesis in hamsters but not in animal models of respiratory tract/lung carcinogenesis, with some suggestions of tumor-promoting effects in respiratory tract/lung. If our results are replicated by other ongoing/completed trials, this suggests a critical need for mechanistic studies addressing differential responses in one epithelial site (head and neck) versus another (lung).

Plasma lycopene concentrations in humans are determined by lycopene intake, plasma cholesterol concentrations and selected demographic factors.

Higher plasma lycopene concentrations have been associated with a reduced risk of several chronic diseases. Determinants of lycopene concentrations in humans have received limited attention. We had blood lycopene concentrations and lycopene consumption data available from 111 participants in a two-center cancer prevention trial involving beta-carotene and examined determinants of plasma lycopene levels cross-sectionally. The median plasma lycopene level was 0.59 micromol/L (range 0.07-1.79). Low plasma concentrations of lycopene were associated with the following variables in univariate analyses: study site (Florida lower than Connecticut, P = 0.001), being nonmarried (P = 0.02), having lower income (P = 0.003), being nonwhite race/ethnicity (P = 0.03), having lower dietary lycopene intake (r = 0.29, P = 0.002), having lower plasma cholesterol (r = 0. 43, P = 0.0001) and triglyceride levels (r = 0.26, P = 0.005), and consuming less vitamin C (r = 0.20, P = 0.03). Women had slightly higher plasma lycopene levels than men (0.65 vs. 0.58 micromol/L; P = 0.31), despite lower dietary intake of lycopene (1,040 vs. 1,320 microg/d; P = 0.50). Plasma lycopene levels did not differ in smokers and nonsmokers. In stepwise regression analyses, the determinants of plasma lycopene were plasma cholesterol, dietary lycopene, and marital status; these three variables explained 26% of the variance in plasma lycopene. Relatively few lifestyle and demographic factors were important determinants of plasma lycopene levels, with plasma cholesterol, marital status, and lycopene intake being of greatest importance.

Effect of supplemental beta-carotene on plasma concentrations of carotenoids, retinol, and alpha-tocopherol in humans.

High doses of beta-carotene, a lipid-soluble nutrient, may affect the plasma concentrations of other lipid-soluble nutrients. The purpose of this study was to assess the effects of long-term daily supplementation with beta-carotene (50 mg/d) on circulating concentrations of other carotenoids, retinol, and alpha-tocopherol over time. Data were available from 259 men and women participating in the Carotene Prevention Trial, a 2-center chemoprevention trial designed to determine whether supplemental beta-carotene can prevent second malignant tumors in patients cured of an early stage cancer of the oral cavity, pharynx, or larynx. Up to 2 blood samples were obtained before the intervention (before and after a 1-mo placebo run-in), with postrandomization samples obtained at 3, 12, 24, 36, 48, and 60 mo. Supplementation with beta-carotene produced a persistent 9- to 10-fold increase in median plasma beta-carotene concentrations (225 nmol/L at baseline to 2255 nmol/L at 3 mo) and a persistent 2-fold increase in median plasma alpha-carotene concentrations (45 nmol/L at baseline to 95 nmol/L at 3 mo). Concentrations of retinol, alpha-tocopherol, lycopene, and lutein/zeaxanthin were not affected by supplemental beta-carotene. Up to 5 y of daily supplementation with beta-carotene increased circulating concentrations of alpha- and beta-carotene, but did not alter concentrations of lycopene, lutein/zeaxanthin, retinol, or alpha-tocopherol.

A prospective, double-blind, randomized study on the use of a topical anesthetic, vasoconstrictor, and placebo during transnasal flexible fiberoptic endoscopy.

The purpose of the present study was to compare patient comfort levels following administration of a topical anesthetic, vasoconstrictor, placebo, or nothing to the nasal mucosa prior to flexible fiberoptic transnasal endoscopy. Using a prospective, double-blind, randomized design, 152 consecutive patients were randomly assigned to receive a topical anesthetic (N = 54), vasoconstrictor (N = 50), or placebo (N = 48). No significant differences were found among the three variables. An additional 50 consecutive patients had endoscopy performed without administration of any substance to the nares, and no significant differences were found among the four variables (N = 202). It was concluded that speech-language pathologists can perform independent and comfortable transnasal endoscopy without administration of any substance to the nasal mucosa. Flexible fiberoptic endoscopy, however, should be performed by experienced clinicians with care taken to examine visually the patency of both nares for ease and comfort of scope insertion.

Apoptotic inhibition of head and neck squamous cell carcinoma cells by tumor necrosis factor alpha.

OBJECTIVES: To evaluate the effect and mechanism of action of tumor necrosis factor alpha (TNF-alpha) on head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, to find out whether TNF-alpha induces apoptotic (programmed) cell death. DESIGN: Cytotoxicity and kinetics were evaluated through the use of a methylene blue colorimetric assay. The mechanism of cell death was evaluated through gel electrophoresis of extracted DNA, double-stranded DNA fragmentation, and Hoechst 33342-propidium iodide double staining. TARGET CELLS: Four HNSCC cell lines were investigated: UMSCC-1, UMSCC-8, UMSCC-19, and CAL-27. RESULTS: Tumor necrosis factor alpha at a concentration of 10000 U/mL induced cytotoxic effects in all four cell lines with varying sensitivities. Significant cytotoxic effects required 3 to 4 days of exposure to TNF-alpha. Further experiments demonstrated that TNF-alpha induced double-stranded DNA fragmentation of targets, which preceded the detection of actual cytotoxic effects. Treated HNSCC cell lines had their DNA fragmented into a ladder pattern of endonucleolytic cleavage of multiples of 180 to 200 base pairs, consistent with apoptosis. Propidium iodide-Hoechst 33342 double staining confirmed that TNF-alpha-induced nuclear alterations precede permeabilization of the plasma membrane, supporting cell death in these tumor cells by apoptosis. CONCLUSION: Tumor necrosis factor alpha exposure results in variable cytotoxic reactions in HNSCC cell lines, with DNA fragmentation preceding significant cell death. The mechanism of cell death is apoptosis, with typical morphological features. Further studies are needed to better elucidate the role of TNF-alpha in the treatment of in vivo HNSCC.

Inhibition of head and neck squamous cell carcinoma cell lines by transforming growth factor-beta.

Transforming growth factor-beta is known to be a potent autocrine growth inhibitor produced by a wide variety of cells, including cells of the immune system. Other investigators have noted that the growth of nontransformed keratinocytes is inhibited by transforming growth factor-beta, whereas various carcinoma cell lines are resistant to these effects. Head and neck squamous cell carcinoma cells are known to have surface receptors for this cytokine. We thus assessed the effect of transforming growth factor-beta on the growth of head and neck squamous cell carcinoma cell lines. Four head and neck squamous cell carcinoma cell lines were incubated with varying concentrations of transforming growth factor-beta, and cytotoxicity was evaluated with a methylene blue colorimetric assay. After culturing in transforming growth factor-beta for 4 days, inhibition of growth was detected in CAL-27 (maximal inhibition at 5.0 ng/ml), UMSCC-1, and UMSCC-19 (maximal inhibition at 50 ng/ml) cell lines. One other cell line, UMSCC-8 was found resistant to the inhibitory effects of transforming growth factor-beta. Kinetics analysis experiments revealed minimal inhibition before day 2 of incubation, at which time inhibition increased linearly to day 4. Assessment of double-stranded DNA fragmentation suggested that DNA fragmentation occurs before significant cytotoxicity. Electron microscopic analysis and gel electrophoresis of extracted DNA revealed morphologic features consistent with apoptotic cell death. Our findings indicate that transforming growth factor-beta significantly inhibits the growth of head and neck squamous cell carcinoma cell lines by inducing apoptotic cell death.

Atypical apoptotic cell death induced in L929 targets by exposure to tumor necrosis factor.

The mechanism by which tumor necrosis factor (TNF) induces cytotoxicity of murine fibroblasts was investigated. Electrophoresis of DNA extracted from TNF-treated L929 targets showed fragmentation of DNA into a ladder-like pattern, typical of cells dying by apoptosis. Morphologic analysis also indicated apoptotic cell death, demonstrating clumping and crescentic condensation of chromatin. In contrast, chromatin condensation and ladder-like DNA fragmentation were not detected in L929 targets dying by necrosis from exposure to heat, repeated cycles of freeze-thaw, and sodium azide. Chromatin condensation was an early event, detected as early as 6 h of incubation. However, DNA fragmentation (assayed by double-stranded fragmentation assay and gel electrophoresis), as well as the apoptotic changes detected by Hoechst fluorescence, both occurred later and did not precede TNF cytotoxicity (membrane permeabilization detected by trypan blue or propidium iodide staining). This atypical pattern of apoptosis was a characteristic of L929 target cells rather than a generalized cytotoxic response to TNF because TNF-treated squamous cancer cells showed typical features of apoptosis (DNA fragmentation before cytotoxicity) and etoposide-treated L929 cells demonstrated the same atypical kinetics as TNF-treated cells. Zinc significantly inhibited TNF cytotoxicity as well as DNA fragmentation of L929. However, because DNA fragmentation occurred belatedly in TNF-treated targets, lagging behind cytotoxicity, the protection by zinc against TNF appears mediated by events that occur before the ultimate endonuclease-induced cleavage of DNA into small fragments.

Inhibition of DNA repair with aphidicolin enhances sensitivity of targets to tumor necrosis factor.

To test whether DNA injury contributes to TNF-induced cytotoxicity, we attempted to enhance DNA injury by inhibiting its repair and then assessing effects on cytotoxicity. DNA repair, assayed as unscheduled DNA synthesis, was first detected in TNF-sensitive targets by 2-3 h of incubation with TNF. Targets resistant to TNF cytotoxicity did not demonstrate significant repair replication. Repair preceded the detection of TNF-induced DNA injury, which was subsequently demonstrated by a double-stranded DNA fragmentation assay, sedimentation of DNA in neutral and alkaline sucrose gradients, and gel electrophoresis of extracted DNA. This suggested that early during exposure to TNF, DNA repair proceeds more rapidly than strand breakage. To inhibit repair, nontoxic concentrations of aphidicolin (inhibitor of DNA polymerase-alpha) and dideoxythymidine (inhibitor of DNA polymerase-beta and gamma) were used. Aphidicolin inhibited repair and consistently sensitized to TNF cytotoxicity, decreasing the ID50 for TNF at least 10- to 50-fold. In contrast, dideoxythymidine had no effect on repair or cytotoxicity. Deoxycytidine, which competitively inhibits binding of aphidicolin to DNA polymerase, blocked the sensitization in a concentration-dependent fashion. In targets sensitized with aphidicolin, TNF-induced strand breakage was accelerated, being detected by 4 h of culture in the sucrose gradient assay. Sensitization to TNF was not due to a heightened activation of poly (ADP-ribose) polymerase. These results indicate that TNF-induced strand breakage participates in TNF-induced cytotoxicity and that the level of DNA repair plays a role in determining relative sensitivity of targets.