tretinoin and Head-and-Neck-Neoplasms

Chemoprevention of cancer is reviewed from the viewpoints of action mechanisms and methodology of clinical trials in order to introduce promising agents discovered by in vitro and/or in vivo studies to applications in humans. The clinical trial procedure essentially follows the phase study which has been employed for chemotherapeutic drugs. Chemoprevention of bladder cancer, prostate cancer, gastric cancer, hepatocellular carcinoma, breast cancer, head and neck cancer, colorectal cancer and lung cancer is reviewed, mainly focusing on clinical trials. Previous clinical trials have shown the effectiveness of the following: polyprenoic acid (acyclic retinoid) for hepatocellular carcinoma; tamoxifen for breast cancer; retinoic acids for head and neck tumor; and aspirin, a COX-2 inhibitor, for colorectal cancer. Despite the advantageous effects of some of these agents, their toxic effects must also be of concern at the same time. For example, in a chemoprevention trial of lung cancer, beta-carotene was unexpectedly found to increase the risk of lung cancer among high-risk groups. It is also noted that large-scale clinical trials demand large research grants, which may not be affordable in Japan. Chemoprevention is still an emerging field of oncology where researchers in both basic and clinical sciences face great challenges.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; beta Carotene; Breast Neoplasms; Clinical Trials as Topic; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Colorectal Neoplasms; Female; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Neoplasms; Prostatic Neoplasms; Tamoxifen; Tretinoin; Urinary Bladder Neoplasms

The ultimate goal of our chemoprevention research is to prevent or inhibit the development of aerodigestive cancer in humans. We have made substantial progress from our trials 10 years ago. The chemopreventive strategies utilized in our clinical trials involve the use of retinoids and carotenoids as chemopreventive agents. The choice of these agents was based upon their important anticarcinogenic and differentiation properties. It is important to understand how retinoids interact with cells to carry out their modulating activities, and we hope to increase our understanding through molecular analysis of retinoid receptors. In the case of aerodigestive epithelial tissues at risk, normal, non-keratinizing epithelial cells often express inappropriate squamous differentiation. Retinoids are thought to suppress premalignant lesions by suppressing these inappropriate squamous differentiation pathways. The role of retinoids in suppressing squamous differentiation markers and reversing premalignant lesions will be elucidated from this retinoid project. The development of a fundamental understanding of tumorigenesis in the aerodigestive tract can lead to novel preventive approaches. A relative degree of risk for cancer development in individuals depends on several components, including the extent of carcinogenic exposure, inherent sensitivity of the individual to carcinogens, the individual's nutritional status, etc. Individuals with a genetic component of increased carcinogen sensitivity appear to be at increased risk for developing primary and secondary tumors. Our chemoprevention research program is designed to develop innovative strategies for aerodigestive tract epithelial cancer prevention. The strength of our program is to bridge the gap between fundamental and cellular molecular studies in clinical chemoprevention trials. The outcome of our research efforts may have an enormous impact on public health in controlling aerodigestive epithelial cancers and other epithelial cancers as well.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Esophageal Neoplasms; Head and Neck Neoplasms; Humans; Lung Neoplasms; Stereoisomerism; Tretinoin

Topics: Animals; Anticarcinogenic Agents; Etretinate; Head and Neck Neoplasms; Humans; Isotretinoin; Retinoids; Tretinoin

Topics: Anticarcinogenic Agents; Combined Modality Therapy; Head and Neck Neoplasms; Humans; Incidence; Laryngeal Neoplasms; Larynx; Precancerous Conditions; Tretinoin; United States

Treatment options are limited for recurrent/metastatic adenoid cystic carcinoma of the head and neck (R/M ACCHN). We aimed to evaluate the preliminary results of the efficacy and safety of all-trans retinoic acid (ATRA) combined with low-dose apatinib in patients with R/M ACCHN according to a secondary analysis of a phase II study.. A total of 16 patients were included with nine (56.3%) males and aged 35-69 years old. All recruited patients previously received anti-angiogenic therapy then withdrew due to toxicities or progression occurred. The objective response rate (ORR) and disease control rate (DCR) were 18.8% and 100%, respectively. During a median follow-up of 23.9 months (range:17.8-31.7 months), 11 (68.8%) patients developed PD and one of them died in 20.9 months. The median of progression-free survival (PFS) was 16.3 months (95% CI: 7.2-25.4 months), and the 6-month, 12-month, and 24-month PFS rates were 100%, 81.3%, and 33.3%, respectively. The grade 3 adverse events were albuminuria (n = 2, 12.5%) and hand-foot syndrome (n = 1, 6.25%).. All-trans retinoic acid combined with low-dose apatinib might be a potential efficacy therapeutic option for patients with R/M ACCHN. This finding will be further confirmed by our registered ongoing trial, the APLUS study (NCT04433169).

Topics: Adult; Aged; Antineoplastic Agents; Carcinoma; Carcinoma, Adenoid Cystic; Female; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Middle Aged; Tretinoin

Although retinoids show promise for prevention of second primary upper aerodigestive tract tumors, the optimum retinoid, dose, and schedule are unknown. All-trans retinoic acid (ATRA) has greater affinity for retinoic acid receptors and may be more active than other retinoids but has a shorter plasma half life and may up-regulate its own metabolism. We defined the maximum long-term tolerable dose, dosing frequency, pharmacokinetics, and toxicity of ATRA in patients with treated squamous cell carcinoma of the head and neck (SCCHN). Twenty-one patients were randomized to 45, 90, or 150 mg/m2 ATRA either once daily, or as divided doses every 8 h, for 1 year. Pharmacokinetics were assessed periodically. Fourteen men and seven women with previous SCCHN of initial stage I-IV were treated. Grade > or =3 toxicities (reversible) included headache and hypertriglyceridemia in 5 and 6 patients each, mucositis in 2 patients, and hyperbilirubinemia, elevated alkaline phosphatase, colitis, lipasemia, xerostomia, eczema, and arthritis in 1 patient each. The 150-mg/m2 dose was not tolerable. Doses were reduced for grade > or =3 toxicity in seven of eight patients at 90 mg/m2 daily. Three of nine patients at 45 mg/m2/day required dose reduction, two at the once-daily dose. Day 1 ATRA area under the plasma concentration versus time curve (AUC) increased with dose, and after 1-2 months of continued dosing, the AUC declined in 7 of 13 patients (54%) studied. ATRA AUC did not correlate with toxicity severity or frequency. Fifteen mg/m2/day every 8 h is a tolerable dose for 1 year in patients with treated SCCHN. ATRA pharmacokinetics did not correlate with toxicity.

Topics: Adult; Aged; Antineoplastic Agents; Area Under Curve; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Exanthema; Female; Follow-Up Studies; Head and Neck Neoplasms; Headache; Humans; Hypertriglyceridemia; Male; Middle Aged; Mouth Mucosa; Neoplasm Staging; Stomatitis; Treatment Outcome; Tretinoin

To achieve locoregional control of head and neck cancer, survival, and organ preservation using intensive concomitant chemoradiotherapy.. This study was a phase II trial of chemoradiotherapy with cisplatin 100 mg/m(2) every 28 days, infusional fluorouracil 800 mg/m(2)/d for 5 days, hydroxyurea 1 g orally every 12 hours for 11 doses, and radiotherapy twice daily at 1.5 Gy/fraction on days 1 through 5 (total dose, 15 Gy). Five days of treatment were followed by 9 days of rest, during which time patients received granulocyte colony-stimulating factor. Five cycles (three with cisplatin) were administered over 10 weeks (total radiotherapy dose, locoregional). Surgery after concomitant chemoradiotherapy is feasible. Compliance with adjuvant chemoprevention is poor. Identification of less toxic regimens and improved distant disease control emerge as important future research goals.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Combined Modality Therapy; Disease Progression; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Hydroxyurea; Interferon alpha-2; Interferon-alpha; Male; Quality of Life; Radiotherapy; Radiotherapy Dosage; Recombinant Proteins; Survival Rate; Tretinoin

Retinoids have been increasingly used since the mid-1960s for the treatment of leukoplakia and dysplasia of the head and neck. Studies of their use in the treatment of carcinomas of the head and neck, usually in combination with interferons, have also been published in recent years.. 30 patients (25 men, five women) with UICC stage IV were given adjunctive treatment with a combination of 3 x 3 million IU of interferon alfa SC weekly and 0.5 mg/kg body weight of 13-cis retinoic acid PO daily for a maximum duration of 6 months. The therapeutic benefits and side effects are reported here.. Therapy was completed as scheduled in 16 out of 30 patients. Reasons for stopping treatment: progressive disease (ten patients), side effects (four patients). 18 patients were tumor-free following treatment. 16 patients displayed a complete response one year after completion of adjunctive treatment. Retinoic-associated side effects observed included xerostomia (90%), dysphagia (67%), weight loss (50%), flush (50%) and cachexia (7%). Interferon-associated side effects included pyrexia and moderate hematological changes.. Adjunctive combination treatment with interferon alfa and 13-cis retinoic acid appears to be beneficial to patients with head and neck cancer. The side effects are moderate.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Combined Modality Therapy; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Interferon-alpha; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Second Primary; Tretinoin

Although tobacco and alcohol use are the major determinants of upper aerodigestive tract carcinogenesis, not all smokers develop cancer. This phenomenon is due to individual variation in genetic susceptibility to carcinogens. One explanation may be differences in mutagen sensitivity (as measured by the in vitro bleomycin-induced mutagen sensitivity assay) in patients with squamous cell carcinoma of the upper aerodigestive tract. Antioxidant supplementation has also been shown to decrease DNA damage and thus may also inhibit carcinogenesis. In this study, we examined whether smoking, alcohol intake, and dietary antioxidant intake were correlated with mutagen sensitivity. The 612 patients evaluated are part of an ongoing multicenter Phase III trial of 13-cis retinoic acid for the prevention of second primary tumors. We found that patients with pharyngeal cancers were more likely than patients with oral cavity or larynx cancers to be mutagen sensitive. There were no significant differences in the distribution of mutagen sensitivity by sex or alcohol use. Never smokers were significantly more likely (61.1%) to be mutagen sensitive than current smokers (35.6%). Dietary consumption of the micronutrients alpha-carotene, beta-carotene, lutein, lycopene, and vitamin C was not correlated with mutagen sensitivity. Therefore, we suggest that mutagen sensitivity is an independent marker of cancer risk not affected by other known risk factors.

Topics: Adult; Aged; Alcohol Drinking; Antineoplastic Agents; Antioxidants; Carcinoma, Squamous Cell; Diet; Female; Genetic Predisposition to Disease; Head and Neck Neoplasms; Humans; Logistic Models; Male; Middle Aged; Mutagenesis; Mutagenicity Tests; Neoplasms, Second Primary; Risk Factors; Smoking; Tretinoin

The ultimate goal of our chemoprevention research is to prevent or inhibit the development of aerodigestive cancer in humans. We have made substantial progress from our trials 10 years ago. The chemopreventive strategies utilized in our clinical trials involve the use of retinoids and carotenoids as chemopreventive agents. The choice of these agents was based upon their important anticarcinogenic and differentiation properties. It is important to understand how retinoids interact with cells to carry out their modulating activities, and we hope to increase our understanding through molecular analysis of retinoid receptors. In the case of aerodigestive epithelial tissues at risk, normal, non-keratinizing epithelial cells often express inappropriate squamous differentiation. Retinoids are thought to suppress premalignant lesions by suppressing these inappropriate squamous differentiation pathways. The role of retinoids in suppressing squamous differentiation markers and reversing premalignant lesions will be elucidated from this retinoid project. The development of a fundamental understanding of tumorigenesis in the aerodigestive tract can lead to novel preventive approaches. A relative degree of risk for cancer development in individuals depends on several components, including the extent of carcinogenic exposure, inherent sensitivity of the individual to carcinogens, the individual's nutritional status, etc. Individuals with a genetic component of increased carcinogen sensitivity appear to be at increased risk for developing primary and secondary tumors. Our chemoprevention research program is designed to develop innovative strategies for aerodigestive tract epithelial cancer prevention. The strength of our program is to bridge the gap between fundamental and cellular molecular studies in clinical chemoprevention trials. The outcome of our research efforts may have an enormous impact on public health in controlling aerodigestive epithelial cancers and other epithelial cancers as well.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Esophageal Neoplasms; Head and Neck Neoplasms; Humans; Lung Neoplasms; Stereoisomerism; Tretinoin

Retinoids, the analogs of vitamin A, are active in vitro and in vivo against squamous cell carcinoma in animals and against certain epithelial precancers and cancers in humans. These data led us to design a prospective, multi-institutional, randomized phase II trial of isotretinoin in advanced head and neck squamous cell carcinoma. We randomly assigned 40 patients to receive isotretinoin or methotrexate, the best-studied and most active single agent for this disease. Overall, the study patients had extremely poor prognoses, i.e., low performance statuses and recurring disease after surgery and/or irradiation. Three objective responses (16%), including one complete response, occurred in the 19 evaluable isotretinoin-treated patients. Only one minor response (5%) occurred in the methotrexate-treated group. Toxicity occurred with both drugs, but was manageable and never life threatening in the retinoid group. These results and the established activity of retinoids in oral leukoplakia (a precursor of head and neck cancer) indicate the need for further study of this class of drugs in head and neck cancer.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Drug Evaluation; Female; Head and Neck Neoplasms; Humans; Isotretinoin; Male; Methotrexate; Middle Aged; Random Allocation; Tretinoin

Ten cats with a total of 15 cancerous or precancerous lesions were examined for clinical response to and histopathologic changes after treatment with 13-cis-retinoic acid. Before treatment was started, the lesions were graded according to clinical severity and biopsied for histopathologic examination. Serum samples were prepared for determining vitamin A concentrations. For comparison, serum vitamin A concentrations in 10 clinically healthy cats were determined. 13-cis-Retinoic acid (approx 3.0 mg/kg) was given to affected cats once a day for an average of 68 days. At the completion of the therapeutic trial, additional biopsy tissues were obtained for histopathologic examination, and serum was assayed for 13-cis-retinoic acid. Of the 15 lesions examined, only 1 showed partial clinical and microscopic improvement during the therapy period. The mean serum vitamin A concentration of the affected cats was not statistically different from that of the 10 healthy cats. The results of this trial indicated that 13-cis-retinoic acid used at this dosage, daily frequency, and duration did not have therapeutic efficacy for squamous cell carcinomas or preneoplastic lesions in the cat and that the mean serum vitamin A concentration did not differ between the affected cats and clinically healthy cats.

Topics: Animals; Carcinoma, Squamous Cell; Cat Diseases; Cats; Clinical Trials as Topic; Head and Neck Neoplasms; Isotretinoin; Precancerous Conditions; Tretinoin; Vitamin A

Cancer stem cells (CSCs), a rare cell population in tumors, are resistant to conventional chemotherapy and thus responsible for tumor recurrence. To screen for active compounds targeting CSCs, a good CSC-enriched model compatible with high-throughput screening (HTS) is needed. Here, we describe a new head and neck cancer stem cell-enriched spheroid model (SCESM) suitable for HTS analyses of anti-CSC compounds. We used FaDu cells, round-bottom ultra-low adherent (ULA) microplates, and stem medium. The formed spheroids displayed increased expression of all stem markers tested (qRT-PCR and protein analysis) in comparison to the FaDu cells grown in a standard adherent culture or in a well-known HTS-compatible multi-cellular tumor spheroid model (MCTS). Consistent with increased stemness of the cells in the spheroid, confocal microscopy detected fast proliferating cells only at the outer rim of the SCESM spheroids, with poorly/non-proliferating cells deeper in. To confirm the sensitivity of our model, we used ATRA treatment, which strongly reduced the expression of selected stem markers. Altogether, we developed a CSC-enriched spheroid model with a simple protocol, a microplate format compatible with multimodal detection systems, and a high detection signal, making it suitable for anti-CSC compounds' HTS.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Models, Biological; Neoplastic Stem Cells; Spheroids, Cellular; Tretinoin

Head and neck cancers (HNC) are known for their repopulation ability driven by cancer stem cells (CSCs). While a small fraction of CSCs proliferates, there are quiescent CSCs that are long-lived and reside outside the cell cycle. Recruitment of quiescent CSCs into the cycle occurs as a response to cell loss and their proliferation may lead to treatment failure. Therefore, CSCs require a more targeted approach to be destroyed. An agent that sensitizes CSC response to treatment is all-trans-retinoic acid (ATRA). The aim of this work is to assess the impact of ATRA combined with radiotherapy on HNC and to analyse the interplay between these agents and cell recruitment.. An in silico model is employed to grow a HNC consisting of all cancer cell lineages, with biologically valid kinetic and dynamic parameters. The fate of both cycling and quiescent cancer stem cells is assessed. The Linear Quadratic model is used to simulate radiotherapy, while cellular recruitment and the effects of ATRA on cancer stem cells are modelled based on literature data.. A Dose Enhancement Factor (DEF) was determined in order to undertake a quantitative assessment of the effect of ATRA on tumour control. Without recruitment, DEF for the tumour population is 1.06, indicating a slight radiosensitizing effect. Yet, when CSCs are being recruited, the dose enhancement factor is significantly greater (DEF = 1.89). Radiation-induced cell arrest and CSC sensitization by ATRA significantly decreases the dose required for CSC eradication in the cycling population. However, the tumour as a whole is not notably affected as the quiescent cells appear to dictate the shape of the survival curve.. The model shows that ATRA exhibits a powerful effect on CSCs when combined with radiotherapy. However, the more radioresistant quiescent cell population should not be ignored, as it can be a potential threat to treatment outcome when cells are recruited into the cell cycle.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Head and Neck Neoplasms; Humans; Neoplastic Stem Cells; Tretinoin

An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far.. Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model.. Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression.. This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Mice, Nude; Neoplasm Transplantation; Phenotype; Prognosis; Proportional Hazards Models; Receptors, Retinoic Acid; Retinal Dehydrogenase; Treatment Outcome; Tretinoin

Retinoic acid (RA) is a critical regulator of cell differentiation, proliferation, and apoptosis in various cell types. Recently, the RA-metabolizing enzyme CYP26A1 (cytochrome P450, family 26, subfamily A, polypeptide 1) has been shown to have an oncogenic function in breast carcinogenesis. However, the relevance of elevated CYP26A1 expression in human cancers remains to be clarified.. We immunohistochemically examined the expression of CYP26A1 in cervical squamous cell carcinoma (SCC) and its precursors, including low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively), as well as head and neck cancer (HNC). The association between CYP26A1 expression and a number of clinicopathological parameters was also evaluated.. CYP26A1 was not expressed in normal cervical epithelium. CYP26A1 expression was present in LSIL but limited to basal and parabasal cells. HSIL cases exhibited strong nuclear expression of CYP26A1 and mixed cytoplasmic expression patterns with widely distributed expression toward the epithelial surface. Importantly, strong cytoplasmic staining of CYP26A1 was observed in 19 of 50 (38%) patients with cervical SCC. Elevated expression of CYP26A1 was significantly associated with younger age (<50 years) and lymph node involvement (pN). Similarly, CYP26A1 was not expressed in non-neoplastic tissues of the head and neck, but strong cytoplasmic staining of CYP26A1 was observed in 52 of 128 (41%) HNC cases. Such strong CYP26A1 expression was significantly associated with the primary tumor stage of carcinomas (pT) and the pathological tumor-node-metastasis (pTNM) stage in HNC.. Our results indicated an elevated CYP26A1 expression in malignant and precancerous dysplastic lesions of the human cervix, which also increased with the progression of cervical squamous neoplasia. In addition, this report is the first to demonstrate the increased expression of CYP26A1 in HNC and its significant correlation with primary tumor growth. These data suggested that CYP26A1 overexpression might contribute to the development and progression of cervical malignancies and squamous neoplasia of the head and neck.

Topics: Carcinoma, Squamous Cell; Cytochrome P-450 Enzyme System; Disease Progression; Female; Head and Neck Neoplasms; Humans; Retinoic Acid 4-Hydroxylase; Tretinoin; Uterine Cervical Neoplasms

There are limited studies on the effects of drugs that modulate epigenetic regulation for head and neck squamous cell carcinoma (HNSCC). This study determined the effect of valproic acid (VPA) on HNSCC.. Growth inhibition effects of VPA alone or in combination with 5-aza-2'deoxycytidine (5-aza-dC) or all-trans retinoic acid (ATRA) was evaluated with MTT and clonogenic assays on 5 HNSCC cell lines. The mechanism of growth inhibition was investigated by looking at markers of terminal differentiation and senescence.. Growth inhibition profiles of HNSCC cell lines varied in response to VPA. Inhibition of clonogenic survival in response to VPA was associated with an upregulation of p21, expression of terminal differentiation markers, and cellular senescence. Notably, a combination treatment of 5-Aza-dC-VPA-ATRA enhanced growth inhibition in cells resistant to VPA.. VPA is a potent inhibitor of proliferation in some HNSCC cell lines, and may be used to treat HNSCC.

Topics: Antineoplastic Agents; Azacitidine; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Decitabine; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Squamous Cell Carcinoma of Head and Neck; Tretinoin; Valproic Acid

Differentiation therapy is a novel approach to eradicate cancer stem cells (CSCs), including head and neck squamous carcinoma CSC (HNSC CSC). All-trans-retinoic acid (ATRA) is a potent differentiating agent. We studied the anti-tumour effect of ATRA on HNSC CSC. HNSC CSCs were differentiated by ATRA in a serum-free conditioned medium. The effect of differentiation on tumour growth was assessed in vitro and in vivo, and chemosensitisation was examined using a colorimetric viability assay. In addition, the involvement of Wnt/β-catenin signalling as an underlying mechanism of the anti-tumour effect of retinoic acid (RA) on HNSC CSCs was assessed. ATRA suppressed the expression of the stem cell markers Oct4, Sox2, Nestin and CD44 in HNSC CSCs and inhibited the proliferation of HNSC CSCs in vitro and in vivo. Furthermore, ATRA treatment augmented the chemosensitising effects of cisplatin. The anti-tumour effects of ATRA may be associated with down-regulation of Wnt/β-catenin signalling. In conclusion, ATRA may be potentially valuable in treatment of HNSC CSC, especially in combination with cisplatin.

Topics: Animals; Antineoplastic Agents; beta Catenin; Carcinoma, Squamous Cell; Cell Proliferation; Cisplatin; Female; Head and Neck Neoplasms; Humans; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Squamous Cell Carcinoma of Head and Neck; Tretinoin; Wnt Signaling Pathway

Topics: Alitretinoin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Cost-Benefit Analysis; Decision Making, Organizational; Dermatologic Agents; Eczema; Evidence-Based Medicine; Federal Government; Hand Dermatoses; Head and Neck Neoplasms; Humans; Internationality; National Health Programs; Neoplasms, Squamous Cell; Peer Review; Quality-Adjusted Life Years; Technology Assessment, Biomedical; Treatment Outcome; Tretinoin; United Kingdom

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Head and Neck Neoplasms; Humans; Neoplasm Proteins; Retinoids; Tretinoin

We investigated the relationship between cell growth and differentiation and COX-2 expression in oral squamous cell carcinoma (SCC) in vitro and in vivo. Treatment of SCC25 oral squamous carcinoma cells with sodium butyrate (SB) at 0.5-5 mM or all-trans retinoic acid (ATRA) at 3-300 microM inhibited cell growth and induced apoptosis in a dose-dependent manner with concomittant increases in expression of keratin 13, p21WAF1/Cip1 and p27Kip1 and decreases in expression of COX-2. These effects were more pronounced with SB than with ATRA. Injection of SB or ATRA near SCC25-derived tumors in nude mice resulted in inhibition of growth and elevation of differentiation of the tumor accompanied by marked keratinization and increased expression of keratin 13 and decreased expression of COX-2. These results show that the differentiation-inducing agents, particularly SB, suppress growth of oral squamous carcinoma cells through apoptosis and induce cell differentiation possibly through mechanisms involving COX-2, p27Kip1 and/or p21WAF1/Cip1 in vitro and in vivo.

Topics: Animals; Apoptosis; Blotting, Western; Butyrates; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase 2; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Isobutyrates; Keratins; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA; Time Factors; Tretinoin; Tumor Suppressor Proteins

The chemotherapeutic effects of all-trans-retinoic acid (atRA) are mediated by the retinoic acid receptor beta (RARbeta), but RARbeta expression is reduced in a number of head and neck carcinoma (HNSCC) cells which causes resistance to RA treatment in half the patients with HNSCC. The possible mechanism for the reduced RARbeta expression has been suggested as the methylation of the CpG islands adjacent to the RA response elements (RARE) in the RARbeta promoter and the loss of histone acetylation. The suppressed RARbeta expression can be reactivated by a demethylating agent (5-aza-2'-deoxycytidine, 5-AzaC) or a histone deacetylase inhibitor (trichostatin A, TSA). Therefore, we sought to determine if the restoration of RARbeta activity, or a combination of these drugs, could restore the sensitivity to RA in RARbeta-negative HNSCC cells with an epigenetically methylated RARbeta promoter region. SqCC/Y1 cells resistant to atRA showed methylated and unmethylated forms in the RARbeta promoter region. RARbeta expression of these cells was restored by 5-AzaC or TSA treatment. Also, treatment with TSA and atRA combined synergistically increased the growth-inhibitory effect and highly induced the transcriptional activation of the RARbeta promoter compared to atRA treatment in HNSCC cells. Additionally, TSA alone and the combination 5-AzaC and TSA increased lysine-9 (Lys-9) acetylation and Lys-4 methylation of the first exon at the RARbeta gene, while decreasing the methylation of Lys-9 in the HNSCC cells.

Topics: Acetylation; Antineoplastic Agents; Azacitidine; Carcinoma, Squamous Cell; Cell Line, Tumor; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lysine; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tretinoin

Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation.. RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates.. Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA.. RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cloning, Molecular; DNA Methylation; DNA Primers; DNA, Neoplasm; Female; Gene Silencing; Head and Neck Neoplasms; Humans; Leukoplakia, Oral; Male; Middle Aged; Molecular Sequence Data; Mouth Neoplasms; Polymerase Chain Reaction; Precancerous Conditions; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tretinoin

Retinoids have shown clinical efficacy in cancer chemoprevention and therapy presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high-throughput Western blotting method (Becton-Dickinson PowerBlot) to evaluate changes in the levels of cellular signaling proteins in head and neck squamous cell carcinoma cells treated with the cytostatic all-trans-retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl)retinamide. Treatments of the head and neck squamous cell carcinoma cells with these retinoids for 24 h resulted in increased levels of 14, 22, and 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The changes in the levels of the following proteins were confirmed by conventional western immunoblotting: all-trans-retinoic acid increased ELF3, topoisomerase II alpha, RB2/p130, RIG-G, and EMAPII and decreased MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O(6)-methylguanine-DNA methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and tomosyn and decreased cathepsin L, Mre11, and topoisomerase II alpha. Some of these proteins were also modulated by these retinoids in other human cancer cell lines. A subset of the proteins were modulated similarly by the different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these retinoids modulate proteins are distinct but may overlap. Some of the retinoid-modulated proteins identified in this study may be novel candidates for mediating different responses to retinoids.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Fenretinide; Head and Neck Neoplasms; Humans; Neoplasm Proteins; Retinoids; Tretinoin

The aim of this study was to confirm if catabolism of all-trans retinoic acid (RA) is enhanced by type I cellular retinoic acid binding protein (CRABP-I) expression and to investigate the effect of this enhanced catabolism on cell proliferation of the head and neck squamous cell carcinoma (HNSCC) cell line, AMC-HN-7. We also analyzed the effects of CRABP-I on RA-induced retinoic acid receptor (RAR) activity. The expression of the CRABP-I in stably transfected AMC-HN-7 cell lines (HN7-BPIa and HN7-BPIb) resulted in a lower sensitivity to administered RA compared with that of controls in a clonogenic assay. HN7-BPIs cells showed an increased amount of polar metabolites of RA in thin-layer chromatography. The transcriptional activity of the reporter plasmid RARE(DR5)-tk-CAT after the treatment of RA was lesser in HN7-BPIs than in controls. These results suggest that the increased CYP26-mediated catabolism of RA by CRABP-I transfection might decrease the amount of RA that is accessible to the nuclear receptors and make HNSCC cells resistant to RA.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Division; Chloramphenicol O-Acetyltransferase; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; Head and Neck Neoplasms; Humans; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin; Tumor Stem Cell Assay

Retinoids can regulate the proliferation and differentiation of various tumor cells. It is thought that nuclear retinoid receptors mediate these effects by regulating gene transcription. The identity of specific retinoid target genes is only beginning to be unraveled. One candidate for mediating retinoid-induced growth suppression is the novel class II tumor suppressor gene tazarotene-induced gene 3 (TIG3). We examined the constitutive and all-trans retinoic acid (ATRA)-inducible expression of TIG3 mRNA in five head and neck squamous cell carcinoma (HNSCC) and five nonsmall cell lung carcinoma (NSCLC) cell lines to determine whether it is associated with their responsiveness to ATRA. The expression patterns of retinoic acid receptor beta (RARbeta), another putative retinoid-inducible tumor suppressor gene, were also examined. The constitutive TIG3 expression was high in one HNSCC cell line and two NSCLC cell lines, and moderate to very low in the other cells. Some RARbeta-expressing cells had either low or undetectable TIG3 levels and vice versa. ATRA (1 microM; 48 h) increased TIG3 mRNA in 4/5 HNSCCs and 3/5 NSCLCs and RARbeta mRNA in some of the same cell lines, but also in cells that did not show TIG3 induction. TIG3 mRNA was induced by ATRA between 6 and 12 h in most of the responsive cells. ATRA concentrations required for TIG3 induction ranged from 1 to 500 nM depending on the cell line. The pan-RAR antagonists AGN193109 and the RARalpha antagonist Ro 41-5253 blocked TIG3 induction by ATRA. ATRA suppressed anchorage-independent colony formation in most cells that had a high or moderate constitutive or induced TIG3 expression level. In contrast, RARbeta mRNA expression pattern was not correlated with sensitivity to ATRA. These results suggest that TIG3 is regulated by ATRA via retinoid receptors in certain aerodigestive tract cancer cells, and its induction by ATRA is associated with the suppression of anchorage-independent growth.

Topics: Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; DNA, Complementary; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Lung Neoplasms; Phenotype; Receptors, Retinoic Acid; RNA, Messenger; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoids play essential roles in the regulation of cell differentiation and in the proliferation of various epithelial tissues, and atRA is one such active metabolite of retinoids. However, despite the known functions of atRA, its clinical applications are limited due to the induced metabolism by the specific cytochrome P-450s in the liver. To overcome the limitation, parenteral administration of atRA-loaded biodegradable microspheres, the PDLLA/PLE microspheres containing atRA, was suggested previously. We evaluated chemotherapeutic efficacy of atRA-loaded microspheres in a human head-and-neck xenograft/nude mouse model. When atRA-loaded microspheres were administered s.c. at 200 mg/kg body weight to athymic nude mice, plasma concentration of atRA could be maintained in a range of 1.2 to 3.7 x 10(-8) M for 4 weeks. As a result, the tumor volume of human head-and-neck cancer was reduced compared to the control group by 51.3% (p < 0.01) at 14 days and by 49.2% (p < 0.05) at 28 days.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Drug Carriers; Drug Delivery Systems; Female; Head and Neck Neoplasms; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microspheres; Neoplasms, Experimental; Survival Rate; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

Vitamin A and D(3) have a very strong differentiation induction effect.. We examined the anti tumor effect on head and neck squamous cell carcinoma (HNSCC) by treatment with several vitamins having strong differentiation induction effects in vitro.. We used KB cell that an oral floor squamous cell carcinoma, vitamins as all-trans retinoic acid (ATRA), 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), 1alpha,25(OH)(2)D(3) (calcitriol) and 22-oxa-1,25-(OH)(2)D(3) (OCT). We determined receptors of vitamin A and D(3) using RT-PCR. Furthermore, we investigated the proliferation of tumor cells in concentration dependency using [3H]TdR uptake method, apoptosis and apoptosis related factors using TUNEL method and real-time PCR, cell cycle changes using flow cytometry, changing of the sensitivity of using MTT method, cytokine production and the angiogenesis factor using ELISA, by treatment with these vitamins.. The deficit of RAR-beta was found in the KB cell. Each vitamin suppressed the cell proliferation, induced apoptosis, and cell cycle arrest, upregulated sensitivity of the chemotherapeutics drugs and downregulated several angiogenesis factors and an apoptotic factor; survivin.. These results support the idea that vitamin A, D(3) and their derivatives are useful for preventing and/or treating patients with HNSCC.

Topics: Angiogenesis Inducing Agents; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Calcitriol; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Cycle; Cell Line, Tumor; Cisplatin; Cytokines; Flow Cytometry; Fluorouracil; Head and Neck Neoplasms; Humans; In Situ Nick-End Labeling; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Vitamin A; Vitamin D

Angiogenesis, an essential step in the development of neoplasia, is a complex process that involves the interaction of tumor cells with stromal cells. Tumor-associated macrophages (TAMs) can participate in the induction of angiogenesis and are of prognostic value in some neoplasms. Specimens from head and neck squamous cell carcinomas (HNSCC) often contain large numbers of TAMs. In addition, experimental evidence has demonstrated that HNSCC tumor cells can attract and activate macrophages to participate in the expression of the angiogenic phenotype. These findings suggest that antiangiogenic therapies for HNSCC must include strategies that will block the recruitment of macrophages into the tumor microenvironment. We investigated the ability of retinoic acid (RA) to modulate the ability of tumor cells to recruit and activate monocytes for participation in tumor angiogenesis. Owing to a decrease in the secretion of MCP-1 and transforming growth factor-beta 1 (TGF-beta 1), tumor cells treated with RA were unable to induce peripheral blood monocyte (PBM) chemotaxis. Also, as a result of the decrease in TGF-beta 1 secretion, RA-treated tumor cells were unable to activate macrophages for secretion of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In addition to its affects on tumor cells, RA also directly altered the ability of monocytes to participate in the tumor angiogenesis process. PBM exposed to RA were unable to migrate toward inducers of PBM such as MCP-1 and TGF-beta 1. Finally, RA decreased the ability of tumor-activated macrophages to secrete IL-8 and VEGF. These data demonstrate alternative mechanisms by which RA may modulate angiogenesis in the tumor microenvironment. In addition, it underscores the necessity to develop antiangiogenic treatment protocols that can block each of the ways in which new blood vessel growth is induced in tumor microenvironments.

Topics: Carcinoma, Squamous Cell; Cell Movement; Endothelial Growth Factors; Head and Neck Neoplasms; Humans; Interleukin-8; Lymphokines; Macrophage Activation; Macrophages; Monocytes; Neovascularization, Pathologic; Phenotype; Tretinoin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Collagen gels are increasingly regarded as reliable scaffolds for studying cells in vitro, displaying the same three-dimensional network of collagen fibers as encountered in vivo. As a contribution to therapeutic control of head-and-neck cancer, we grew HSCO86 cells in collagen gel and assessed their behavior in the presence of retinoic acid (RA) and radiation.. The malignant epithelial cell line HSCO86 was isolated from a postirradiation human oropharyngeal squamous carcinoma; it was EGFR-negative by immunocytochemical criteria. The cells were embedded in hydrated collagen I at a density of 10(6) cells/mL, and on Days 8, 10, and 12 of culture, they were treated with 10(-5) M retinoic acid. Radiation was administered using two different schedules: simultaneously with RA in three daily doses totaling 10 Gy, or with a single dose of 8 Gy on Day 29 of culture, after the effects of RA had taken place. Cell proliferation was evaluated by the MTT assay, whereas morphometric characteristics were detected in the cultured gels directly or in the gels after they were fixed and stained with hematoxylin.. Contrary to growth in monolayer, where HSCO86 cells displayed a high proliferation rate, in collagen gel only a tiny fraction of the cells, usually less than 0.02%, survived the environmental stress; these cells spontaneously organized themselves into clonal multicellular spheroids growing up to 0.8 mm in diameter. After exposure to 10(-5) M retinoic acid, cell proliferation first declined and then, about 15 days after treatment, it started to increase to a level far above that in the control group. This surge in proliferation was ascribed to the appearance of numerous fibroblast-like cells at the edge of the spheroids. These cells, called HSCO-F, were the result of epithelial-to-mesenchymal conversion. When the gels were disaggregated by collagenase, and the cells were seeded in monolayer, HSCO-F cells reversed their morphology into parental HSCO86 cells. Treatment of collagen gels with 10 Gy, fractionated in three daily doses, did not substantially affect the growth of HSCO86 spheroids. However, when radiation was given simultaneously with RA, cell growth was significantly inhibited, both in terms of cell proliferation and size of spheroids (p < 0.0001 vs. untreated controls). This synergism applied mainly to parental HSCO86 cells, because no significant damage was induced by radiation on the HSCO-F cells previously generated by treatment with RA.. Differences in the radiosensitivity of HSCO86 and HSCO-F cells are surprising in view of their common origin; this suggests a scenario in which, to overcome a microenvironmental stress, head-and-neck carcinoma cells can temporarily shift from an epithelial to a mesenchymal phenotype. In particular, morphologic and functional data suggested that HSCO-F cells were transformed into vascular endothelial cells whose characteristics included the following: (1) distinctive expression of Factor VIII and beta(1)-integrin, not detected in parental HSCO86 cells; (2) active migration in the collagen network by extruded pseudopodia, frequently appearing as colonies of filamentous cells aligned along the radial axis of the spheroids; and (3) efficient contraction of floating collagen gels. The implication of our study is that head-and-neck carcinomas may respond to RA treatment by selecting cell populations both resistant to radiation and capable of migrating inside the connective tissue, mimicking the behavior of vascular capillaries.

Topics: Cell Division; Collagen; Dose-Response Relationship, Drug; Endothelium, Vascular; Epithelial Cells; Gels; Head and Neck Neoplasms; Humans; Hyaluronic Acid; Immunohistochemistry; Phenotype; Tetrazolium Salts; Thiazoles; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). It has been shown that metabolic pathways of retinoids are important in their anticancer effect and that these pathways may change during HNSCC carcinogenesis. We have previously reported that HNSCC cells have a 17-fold greater turnover rate of retinoic acid (RA) than normal oral keratinocytes from noncancer controls, and that the formation of polar metabolites such as 4-oxo-RA and 4-hydroxy-RA is only seen in HNSCC cell lines. We aimed to establish whether this altered retinoid metabolism is an intrinsic characteristic of HNSCC patients.. The normal mucosa of cancer and noncancer patients was the source of keratinocyte cultures. The cells were exposed to RA for various time periods, and the levels of various retinoids were measured in the culture medium and cell pellets with reverse-phase liquid chromatography.. Cells from cancer patients were morphologically normal and showed no genetic aberrations (i.e. loss of heterozygosity). The RA turnover rate in normal oral keratinocytes of cancer patients was 15 times higher (p = 0.003) than that in normal oral keratinocytes of noncancer controls, with average turnover rates of 218.6 and 14.8 pmol/mg protein/h, respectively. Specific profiles of RA metabolites were similar.. The observed higher RA metabolism in noncancer cells of HNSCC patients suggests that individuals with a relatively high RA turnover have an increased risk of developing HNSCC.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cells, Cultured; Female; Head and Neck Neoplasms; Humans; Keratinocytes; Loss of Heterozygosity; Male; Middle Aged; Mouth; Tretinoin

All-trans retinoic acid (RA) can be catabolized to polar metabolites by microsomal P450s (P450). The aim of this study was to confirm if retinoic acid 4-hydroxylase (CYP26) is a P450 induced by RA and to investigate the role of cellular RA binding proteins (CRABPs), using a slow catabolizer, AMC-HN-4, and a rapid catabolizer, AMC-HN-6. Also, we analyzed the effect of RA catabolism on cell proliferation of head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Both cell lines weakly expressed CYP26 and CRABPs, but RA induced CYP26 only in AMC-HN-6. The sensitivity to RA was variable by the amount of CYP26, and the rapid catabolism by CYP26 made AMC-HN-6 resistant to RA in vitro. In addition, The RA had a stronger effect on the inhibition of tumor growth of AMC-HN-4 than that of AMC-HN-6 in vivo. Conclusively, the CYP26 activity might be one essential factor for the RA sensitivity, but in cells showing induction of CYP26, the RA sensitivity is inversely related to the rate of RA catabolism.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Chromatography, High Pressure Liquid; Culture Media; Cytochrome P-450 Enzyme System; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Kinetics; Mice; Mice, Nude; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

We have used a combination of vitamin A (all-trans-retinyl palmitate), 5-fluorouracil (5-FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiotherapy is called "FAR therapy." In this study we examined the effects of all-trans-retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5-FU on two HNSCC cell lines (YCU-N861 and YCU-H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 mM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused G1 cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27(Kip1) and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU-N861 cells, ATRA also caused a decrease in Bcl-2 and Bcl-X(L) and an increase in Bax. Both ATRA and 5-FU activated c-Jun N-terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU-H891 cells, in which the epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5-FU and the combination of both agents than YCU-N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5-FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Division; Cell Survival; Cyclin D1; DNA-Binding Proteins; Drug Synergism; ErbB Receptors; Flow Cytometry; Fluorouracil; Head and Neck Neoplasms; Humans; Immunoblotting; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). Previous studies have shown that the metabolic pathways of retinoids are important in the anticancer effect of retinoids, and that these pathways may change during carcinogenesis. In the present study, we analyzed HNSCC cell lines (n = 11) and normal oral keratinocyte cultures (n = 11) by reverse-phase high-performance liquid chromatography and conducted growth inhibition assays. We demonstrate here that in contrast to normal oral keratinocytes, HNSCC cell lines: (a) had averaged a 17-fold greater turnover rate of all-trans-retinoic acid (RA); (b) had a 1.9-fold less RA-induced growth inhibition; (c) were able to form polar metabolites; and (d) were able to catabolize 4-oxo-RA. Furthermore, the mRNA expression of the RA-specific 4-hydroxylase, CYP26A1, was dramatically increased after RA-induction in the two HNSCC cell lines with the highest metabolism, was undetectable in normal keratinocytes, and was not inducible by RA. Next, introduction of CYP26A1 cDNA in a low-metabolizing HNSCC cell line resulted in an 11-fold higher turnover rate of RA and a 12-fold increase in the amount of polar metabolites, but it did not change sensitivity to RA. These observations point to fundamental changes in RA metabolism pathways during HNSCC carcinogenesis and may provide clues to a more rational approach for RA-mediated intervention.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cytochrome P-450 Enzyme System; Head and Neck Neoplasms; Humans; Keratinocytes; Mixed Function Oxygenases; Mouth; Retinoic Acid 4-Hydroxylase; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids, analogues of vitamin A, can reverse premalignant lesions and prevent second primary tumors in patients with head and neck squamous cell carcinoma (HNSCC). The effects of retinoids are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which act as ligand-activated transcription factors. The regulation of cell growth, differentiation and retinoid metabolism in normal, premalignant and malignant cells by retinoids is thought to be a result of their effects on gene expression. We investigated mRNA expression of RARs (alpha, beta, and gamma) and RXR-beta by means of RNase protection and related this to retinoic acid (RA)-induced growth inhibition and RA turnover in four HNSCC cell lines (UM-SCC-14C, UM-SCC-22A, UM-SCC-35 and VU-SCC-OE). An RA-resistant subline of UM-SCC-35 was generated by exposure to increasing concentrations of RA for 8 months (designated UM-SCC-35R). RA turnover was determined on the basis of decreasing RA levels in the cells and culture medium after exposure to 1 microM RA. We found that RAR-gamma mRNA expression was strongly correlated with RA-induced growth inhibition (p = 0.016, R = 0.92) and RA turnover (p = 0.041, R = 0.86). RAR-beta transcript levels were reduced in three of five cell lines compared with normal mucosa, and these did not correlate with RA-induced growth inhibition and RA turnover. Expression of RAR-alpha and RXR-beta was not substantially altered in any of the cell lines. These findings suggest that in HNSCC cell lines RAR-gamma is the most important retinoid receptor for regulation of RA turnover rate and RA-induced growth inhibition.

Topics: Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4HPR), which is currently being evaluated in clinical trials for cancer prevention and therapy, inhibits the growth of a variety of malignant cells through induction of apoptosis. However, in the majority of tumor cells, this inhibitory effect of 4HPR requires high concentrations (>1 microM), which exceed the peak plasma level measured in humans. In the present study, we compared and contrasted the effects of several synthetic retinamides on the growth of human lung and head and neck cancer cells in vitro. We found that some retinamides, especially N-(2-carboxyphenyl)retinamide (2CPR), exhibited better growth inhibitory effects than 4HPR in some of the cell lines. 2CPR exerted potent growth inhibitory effects in 5 of 10 head and neck cancer cell lines and in 1 of 10 lung cancer cell lines (IC(50), <0.8 microM). 2CPR (1 microM) induced apoptosis ranging from 10 to 60% in four of five cell lines, whereas 4HPR was ineffective at the same concentration. Unlike 4HPR, 2CPR (up to 10 microM) failed to induce reactive oxygen species production in these sensitive cell lines but could activate caspases 3 and 7 as well as increase poly(ADP-ribose)polymerase cleavage. Interestingly, the effect of 2CPR on cell growth could be suppressed by the specific retinoic acid receptor pan antagonist AGN193109. Our results suggest that 2CPR acts via retinoic acid receptors and may be a good candidate for prevention and treatment of some head and neck and lung cancers.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Fenretinide; Head and Neck Neoplasms; Humans; Lung Neoplasms; Reactive Oxygen Species; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured

Adenovirus-mediated p53 (AdCMVp53) gene therapy for cancer is currently undergoing phase III clinical trials. One problematic aspect of this therapy is that the current protocols result in low transduction of the therapeutic virus in vivo. To search new modalities that can enhance the effect of AdCMVp53 gene therapy, we focused on retinoids.. To study the effect of ATRA in combination with AdCMVp53 gene therapy, we pretreated head and neck squamous cell carcinoma (HNSCC) cells for 72 hours with a low-dose All-trans-retinoic acid (ATRA) (10-7 M-10-8 M) which will not affect the in vitro cell growth, and then infected the cells with low MOI (30MOI) AdCMVp53. In vitro cell proliferation assays, cell cycle assays were performed. Expression of p53 and p53-related gene products, BAX and p21, were examined.. The combined treatment with ATRA and Ad-p53 suppressed cell growth and induced apoptosis significantly more than AdCMVp53 treatment alone (P <.05). p53 expression significantly increased more after the combined treatment than after either treatment alone, at both the transcription and protein levels. In addition, increased expression of p21 and BAX, which are downstream gene products of p53, was observed in the combination. ATRA also enhanced the expression of green fluorescent protein (GFP) transduced by an adenovirus-cytomegalovirus (CMV)-GFP vector suggesting ATRA enhances adenovirus-CMV-promoted vectors through transcription.. Our results indicate that ATRA enhances AdCMVp53 expression through transcriptional mechanisms and can synergistically induce apoptosis in HNSCC cells. ATRA has a potential to enhance the effect of adenovirus-mediated p53 gene therapy for HNSCC.

Topics: Adenoviridae; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Genes, p53; Genetic Therapy; Head and Neck Neoplasms; Humans; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoids' effects on cell growth and differentiation are mediated by nuclear retinoid receptors, which are ligand-activated transcription enhancing factors. Because the expression of the retinoic acid receptor beta (RARbeta) gene, which is located on chromosome 3p24, is diminished in premalignant and malignant tissues it has been proposed that it acts as a tumor suppressor. To test the hypothesis that RARbeta loss leads to retinoid resistance, we studied several karyotyped head and neck squamous carcinoma (HNSCC) cell lines (UMSCC-17A, -17B, -22A, -22B, and -38) with deletion of one chromosome 3p arm. RARbeta mRNA was neither detected nor induced by retinoic acid in these cells, whereas it was expressed and induced by retinoic acid in two other HNSCC cell lines (1483 and 183) without 3p deletion. Methylation of the RARbeta gene promoter was detected in the 17B and 22B cells that failed to express RARbeta but no methylation was found in 183A cells that did express RARbeta mRNA. Responsiveness of HNSCC cells to several retinoids in assays of growth inhibition and colony formation, was rank ordered as: 22B>1483>38>183>17B. Additionally, retinoid response elements were transactivated in 22B more efficiently than in 17B cells. These results indicate that loss of RARbeta expression does not necessarily lead to loss of growth inhibition by retinoids or to a block of retinoid signaling.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Chromosome Deletion; Chromosomes, Human, Pair 3; DNA, Neoplasm; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoids; RNA, Neoplasm; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Tumor neovascularization is necessary for the progressive development of all solid tumors, including head and neck squamous cell carcinomas (HNSCCs). The angiogenic process includes increased endothelial cell motility. Our prior studies have shown the importance of protein phosphatase-2A (PP-2A) in restricting endothelial cell motility. Because motility is regulated by the polymerization/depolymerization of the cellular cytoskeleton, the present study defined the interrelationship between PP-2A and the cytoskeleton during endothelial cell responses to HNSCC-derived angiogenic factors. PP-2A was shown to colocalize with microtubules of unstimulated endothelial cells. However, exposure to HNSCC-derived products resulted in a more diffuse distribution of PP-2A staining and a loss of filamentous tubulin. The feasibility of pharmacologically preventing this cytoskeletal disorganization as a means of blocking tumor-induced angiogenesis was tested. This was accomplished by use of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and all-trans -retinoic acid to indirectly stimulate PP-2A activity through their capacity to elevated intracellular levels of the second messenger ceramide. Pretreatment of endothelial cells with either 1,25(OH)(2)D(3) or retinoic acid prevented the cytoskeletal disorganization that otherwise occurs in endothelial cells on exposure to HNSCC-derived products. These studies support the feasibility of using elevation of PP-2A to prevent the morphogenic component of the angiogenic process that is stimulated by HNSCC-derived factors.

Topics: Angiogenesis Inducing Agents; Blotting, Western; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cell Movement; Culture Media, Conditioned; Cytoskeleton; Endothelium, Vascular; Enzyme Activation; Head and Neck Neoplasms; Humans; Immunohistochemistry; Microtubules; Neovascularization, Pathologic; Phosphoprotein Phosphatases; Protein Phosphatase 2; Tretinoin; Tubulin; Tumor Cells, Cultured

The preventive effects of retinoids on oral carcinogenesis may be related to their ability to modulate the growth and differentiation of human oral squamous epithelial cells. Nuclear retinoid receptors (RAR alpha, beta, and gamma, and RXR alpha, beta, and gamma) may mediate these effects by regulating gene transcription. The removal of serum from the growth medium of two head and neck squamous cell carcinoma lines 1483 and SqCC/Y1 resulted in a decrease in RAR beta mRNA level and concurrent increases in the expression of the keratin K1 and transglutaminase type I (TGase I), which are markers of differentiation of keratinizing squamous epithelial cells. All-trans-retinoic acid (tRA) or 13-cis-RA increased RAR beta and decreased K1 and TGase I mRNA levels in serum-free medium. Transcriptional activation of reporter genes by means of retinoid response elements (RARE and RXRE) indicated that the RXR-RAR pathway predominates over the RXR homodimer pathway in the 1483 cells. Among several synthetic retinoids with preference for binding to specific nuclear retinoid receptors, those that induced RAR beta also suppressed K1. The inverse association between RAR beta expression and K1 and TGase I levels implicates this receptor in suppression of keratinization in oral epithelial cells.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Collagenases; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Keratins; Promoter Regions, Genetic; Receptors, Retinoic Acid; Response Elements; Retinoid X Receptors; Retinoids; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Nuclear retinoic acid receptor beta(RARbeta) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARbeta expression in such cells. To investigate the role of RARbeta in HNSCC growth and differentiation, we transfected a retroviral RARbeta2 expression vector (LNSbeta) into HNSCC SqCC/Y1 cells, which do not express endogenous RARbeta but do express RARalpha, RARgamma, and retinoid X receptors. Transfected clones expressing RARbeta2 mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10(-10) M ATRA in four of five LNSbeta clones, whereas it was not suppressed in LNSX cells even by 10(-6) M ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSbeta clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSbeta-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale necarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARbeta mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.

Topics: 3T3 Cells; Alitretinoin; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Drug Resistance; Head and Neck Neoplasms; Humans; Mice; Naphthalenes; Neoplasm Proteins; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; RNA, Neoplasm; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

Products of arachidonic acid metabolism can influence normal and malignant cell growth. In vivo, inhibitors of arachidonic acid metabolism have been associated with inhibition of tumor growth, including head and neck squamous cell carcinoma (HNSCC). This has not been evaluated extensively in vitro in an HNSCC model. Therefore we investigated the effects of several arachidonic acid cascade inhibitors (AACIs) (indomethacin, curcumin, phenidone, nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, and 13-cisretinoic acid) on the growth of two HNSCC cell lines (MDA 886Ln and 1483). We found that AACIs caused dose-dependent growth inhibition of both cell lines. In an effort to inhibit HNSCC cell growth at lower concentrations of these drugs, we evaluated the effects of a variety of AACIs in combination with 13-cis retinoic acid. We observed synergistic growth inhibition when the drugs were used in all combinations, with the exception of indomethacin. These results suggest that AACIs may have some utility in the direct treatment of HNSCC, and a strategy combining 13-cis retinoic acid with other AACIs may prove to be even more effective.

Topics: Arachidonic Acid; Carcinoma, Squamous Cell; Cell Division; Cyclooxygenase Inhibitors; Drug Synergism; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Prostaglandins E; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (RA) can be catabolized into polar metabolites by cytochrome P450 (P450) in several tissues including the skin. We examined eight different squamous cell carcinoma (SCC) cell lines to determine their capacity to induce P450-mediated oxidation of RA. Among the eight different cell lines, enhanced catabolism was detected in AMC-HN-1, -2, -5, and -6, whereas it was not found in the cell lines of AMC-HN-3, -4, -7, and -8. It was found that the enhanced catabolism brought on by P450 induction was blocked when RA was added to AMC-HN-6 along with actinomycin D or cyclohexamide. Also, this catabolism was inhibited by ketoconazole. P450-mediated oxidation was detectable within 4 hours of RA treatment, and RA catabolism reached its maximum 16 hours after treatment. P450 was induced by 13-cis-RA, 9-cis-RA, and retinal; however, retinol could not induce P450. In conclusion, P450 can be induced by retinoids in head and neck SCC (HNSCC) cells and the ability of retinoids to induce P450 can serve as an important factor in determining the biological effect of retinoids.

Topics: Antibiotics, Antineoplastic; Antifungal Agents; Antineoplastic Agents; Carcinoma, Squamous Cell; Cycloheximide; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dactinomycin; Enzyme Induction; Head and Neck Neoplasms; Humans; Ketoconazole; Oxidation-Reduction; Protein Synthesis Inhibitors; Retinoids; Tretinoin; Tumor Cells, Cultured

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy in which multiple independent lesions develop over time throughout the mucosa of the upper aerodigestive tract. Therefore, the comprehensive treatment of this neoplasm must include a chemopreventive arm to hold premalignant lesions in check, a role well-suited to antiangiogenic agents. Retinoic acid (RA) and interferon alpha (IFN-alpha), drugs with known biological activity against HNSCC when used individually, are also inhibitors of angiogenesis. Here we show that they are remarkably synergistic antiangiogenic agents able to inhibit both the growth and the neovascularization of HNSCC injected into the floor of the mouth of nude mice. The mechanism of action of these drugs as antiangiogenic agents was 2-fold. They decreased the angiogenic activity of the tumor cells, and they caused the endothelial cells to become refractory to inducers of angiogenesis. When tumor cells were treated in vitro with IFN-alpha A/D, there was a dramatic drop in their secretion of interleukin-8, the major angiogenic factor produced by these tumors. When combined with RA, which causes tumor cells to secrete an inhibitor of angiogenesis, there was a synergistic inhibition of both tumor cell growth and secreted angiogenic activity. The combination of RA and IFN-alpha also acted synergistically on endothelial cells by reducing their responsiveness to both interleukin-8 and tumor conditioned media. Doses of each drug could be reduced by two logs without loss of activity. When animals bearing human HNSCC tumor cells were treated systemically with a combination of RA and IFN-alpha A/D at doses that were ineffective when used alone, dramatic decreases in both tumor growth and tumor angiogenesis were seen. These data suggest that the use of antiangiogenic mixtures may be a particularly effective way to design future chemoprevention protocols against HNSCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cattle; Cell Division; Cells, Cultured; Chemoprevention; Drug Synergism; Endothelium, Vascular; Female; Head and Neck Neoplasms; Humans; Interferon Type I; Mice; Mice, Nude; Neovascularization, Pathologic; Rats; Rats, Inbred F344; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

The effects of retinoids such as all-trans-retinoic acid (ATRA) on cell growth, differentiation, and apoptosis are thought to be mediated by nuclear retinoid receptors, which are involved in ligand-dependent transcriptional activation of target genes. Using differential display, we identified the cDNA of a novel gene, designated retinoic acid-inducible gene 1 (RAIG1), which was induced by ATRA in the squamous carcinoma cell line UMSCC-22B. Two RAIG1 transcripts of 2.4 and 6.8 kilobase pairs, respectively, have the same ORF that encodes a 357-amino acid polypeptide. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid (within 2 h) and dose-dependent in the range between 1 nM to 1 microM. The constitutive RAIG1 mRNA levels, which were low in three of five head and neck and four of six lung cancer cell lines, increased after ATRA treatment in most cell lines. The deduced RAIG1 protein sequence contains seven transmembrane domains, characteristic of G protein-coupled receptors. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. RAIG1 was mapped to chromosome 12p12. 3-p13. Our results provide novel evidence for a possible interaction between retinoid and G protein signaling pathways.

Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Squamous Cell; Cell Compartmentation; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation; GTP-Binding Proteins; Head and Neck Neoplasms; Humans; Lung Neoplasms; Molecular Sequence Data; Multigene Family; Neoplasm Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Sequence Analysis, DNA; Signal Transduction; Tretinoin; Tumor Cells, Cultured

To understand the role of retinoids in chemoprevention, the authors examined the effects of 13-cis retinoic acid (cRA) on squamous cell carcinoma proliferation and adhesion to extracellular matrix proteins. The antiproliferative effects of cRA were first seen on day 11 (66% inhibition) and progressed through day 19 (96% inhibition). Using an adhesion assay, the authors then investigated the effects of cRA and transforming growth factor-beta1 (TGF-beta1) on cellular adhesion to purified type IV collagen, fibronectin, and laminin matrices. Cells treated for 4 days with TGF-beta1 increased adhesion by 15% to 29%, and cells treated with cRA increased adhesion by 19% to 39%. However, the use of cRA alone resulted in a decrease in adhesion when tumor cells were treated for 7 days (20% to 32%) and 15 days (25% to 40%). The authors also discuss how cRA acts as a differentiating agent on squamous cell carcinoma.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Division; Drug Screening Assays, Antitumor; Extracellular Matrix Proteins; Head and Neck Neoplasms; Humans; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoids can reverse potentially premalignant lesions and prevent second primary tumours in patients with head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been reported that acquired resistance to all-trans retinoic acid (RA) in leukaemia is associated with decreased plasma peak levels, probably the result of enhanced retinoid metabolism. The aim of this study was to investigate the metabolism of retinoids and relate this to growth inhibition in HNSCC. Three HNSCC cell lines were selected on the basis of a large variation in the all-trans RA-induced growth inhibition. Cells were exposed to 9.5 nM (radioactive) for 4 and 24 h, and to 1 and 10 microM (nonradioactive) all-trans RA for 4, 24, 48 and 72 h, and medium and cells were analysed for retinoid metabolites. At all concentrations studied, the amount of growth inhibition was proportional to the extent at which all-trans-, 13- and 9-cis RA disappeared from the medium as well as from the cells. This turnover process coincided with the formation of a group of as yet unidentified polar retinoid metabolites. The level of mRNA of cellular RA-binding protein II (CRABP-II), involved in retinoid homeostasis, was inversely proportional to growth inhibition. These findings indicate that for HNSCC retinoid metabolism may be associated with growth inhibition.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Chromatography, High Pressure Liquid; Culture Media; Head and Neck Neoplasms; Humans; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured

Retinoids are natural and synthetic analogues of vitamin A and have proven activity in various types of cancer. As for head and neck squamous cell cancer (HNSCC), retinoids are especially active in leukoplakia and in preventing second primary cancers. The aim of this study was to assess the growth inhibiting activity of all-trans retinoic acid (all-trans RA) in a panel of six head and neck squamous cell cancer cell lines and to correlate this response to the mRNA expression of factors related to differentiation and receptor mediated signal transduction. Three lines showed minimal, two moderate and one strong growth inhibition after 72 h exposure to all-trans RA. Three lines with a dissimilar response were selected for further studies, the measurement of mRNA expression by northern blotting. It was found that neither the expression nor the induction of retinoic acid receptor (RAR)-alpha and -gamma and retinoic X receptor-alpha mRNA war related to sensitivity. The mRNA expression of RAR-beta was too low to be measured in the three cell lines. The most sensitive cell line was, however, the only one that expressed mRNA of squamous differentiation markers. These data suggest a relationship between the retinoid sensitivity profile and the degree of cellular differentiation.

Topics: Carcinoma, Squamous Cell; Cell Division; Cornified Envelope Proline-Rich Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Keratins; Membrane Proteins; Neoplasm Proteins; Proteins; Receptors, Retinoic Acid; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Cisplatin exerts its cytotoxicity by inducing apoptosis. Similarly, all-trans retinoic acid (ATRA) causes apoptosis in certain cells. We studied the interaction of cisplatin and ATRA in human ovarian adenocarcinoma cells 2008, in human head and neck squamous carcinoma cells UMSCC10b, and in their respective cisplatin-resistant sub-lines. ATRA enhanced the cytotoxicity of cisplatin. The interaction of the drugs was synergistic in combination index-isobologram analyses (combination index >0.5 at 50% cell survival) in all of the cell lines tested. ATRA inhibited the cellular accumulation of the cisplatin analogue [3H] cis-dichloroethylenediamineplatinum(II) by 22-33% in three of four cell lines tested but did not alter the cellular content of reduced glutathione. The expression of Bcl-2 relative to Bax decreased more after combined treatment with cisplatin and ATRA than after either drug alone. The apoptotic mechanism of cell death was confirmed by demonstrating cleavage of poly(ADP-ribose)polymerase and by morphological analysis. The combined treatment with ATRA and cisplatin induced apoptosis in significantly more cells than either drug alone. We conclude that ATRA enhances the cytotoxicity of cisplatin by facilitating apoptosis in ovarian and head and neck carcinoma cells.

Topics: Adenocarcinoma; Apoptosis; Carcinoma, Squamous Cell; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; Female; Head and Neck Neoplasms; Humans; Kinetics; Ovarian Neoplasms; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) has been shown to be effective in eradicating premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck (SCCHN) in clinical trials. The basis for this effect is unclear. We have previously demonstrated that messenger RNA from tumor growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGFR), is unregulated in tumors and histologically normal mucosal samples from patients with SCCHN compared with control normal mucosa from patients without cancer, implicating this ligand-receptor pair in an autocrine growth pathway early in the molecular pathogenesis of this disease. In this report, we examined the hypothesis that the action of RA on the mucosa of the upper aerodigestive tract is mediated via downregulation of steady-state TGF-alpha and/or EGFR mRNA levels. Following exposure to all-trans-RA, a series of SCCHN cell lines demonstrated a 35.4% reduction in TGF-alpha mRNA expression (P = 0.022) and 58.5% reduction in EGFR mRNA (P = 0.0027). Nuclear run-on analysis indicated that the RA-mediated reduction of TGF-alpha and EGFR steady-state mRNA levels was a result of decreased gene transcription. These results suggest that the clinical effects of RA in SCCHN patients may be due to a downmodulation of TGF-alpha and EGFR mRNA production.

Topics: Carcinoma, Squamous Cell; Down-Regulation; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Keratolytic Agents; Mouth Mucosa; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) modulates the growth and differentiation of various normal and malignant cells. These effects are most likely mediated by changes in gene expression. Genes whose expression is modulated by RA may be useful as markers of growth responsiveness to retinoids. Using differential cDNA cloning we identified 10 genes regulated by RA in the head and neck squamous cell carcinoma cell line MDA886Ln. Keratin (K) 13 gene expression was the gene expression most related to the degree of sensitivity of growth to RA, as K13 was not expressed in a series of RA-resistant cell lines. Our data suggest that low K13 expression may be mechanistically related to resistance to RA-induced growth inhibition.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA, Complementary; Epithelium; Gene Expression; Head and Neck Neoplasms; Humans; Keratins; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Nuclear retinoic acid receptors are considered to be the mediators of most of the effects of retinoic acid (RA) on gene expression. To explore the role of RA receptor gamma (RARgamma) in the growth and differentiation of SqCC/Y1 head and neck squamous carcinoma cells, they were transfected with RARgamma sense and antisense expression vectors and stable clones in which RARgamma expression was either increased or blocked were isolated. The growth inhibitory effect of RA in monolayer culture was enhanced in the sense transfectants and decreased in the antisense ones. The ability to form colonies in semisolid medium was abolished by RA in the sense transfectants, while the antisense transfected clones exhibited heterogeneous responses. The expression the squamous differentiation markers cytokeratin K1 transglutaminase type I, and involucrin was increased in the absence of exogenous retinoid in a sense transfected clone and decreased in an antisense transfected clone. RA suppressed squamous differentiation in both types of transfectant. The expression of epidermal growth factor receptor (EGFR) was higher in the antisense and lower in the sense transfectant than in the parental cells and RA decreased EGFR mRNA level in the parental and the sense transfectant but not in the antisense transfectant. In addition activator protein-1 (AP-1) binding activity was decreased by the RA treatment in the sense clones, but not in the antisense ones. These results suggest that RARgamma mediates the effects of RA on the cell growth both in monolayer culture and in semisolid medium possibly through AP-1 suppression.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; ErbB Receptors; Head and Neck Neoplasms; Humans; Molecular Sequence Data; Oligonucleotides, Antisense; Protein Binding; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

The serum concentrations of all-trans (atRA) and 13-cis (13cRA) retinoic acid were determined by high performance liquid chromatography in 27 patients with squamous cell carcinoma of the head and neck and in 80 healthy controls. This investigation seemed relevant as ethanol is an aetiological factor in these cancers and has been suggested to interfere with the synthesis of atRA. Neither the serum concentration of atRA nor that of 13cRA differed between patients and controls. The serum atRA concentration did not differ between fasting and non-fasting patients, but the serum 13cRA concentration was significantly higher in non-fasting than in fasting patients, probably due to the dietary retinoid content.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Chromatography, High Pressure Liquid; Fasting; Female; Head and Neck Neoplasms; Humans; Isotretinoin; Male; Middle Aged; Tretinoin

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of beta-all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 microM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [3H]-glucosamine, [3H]-galactose, and [3H]-mannose into numerous cellular glycoconjugates, however, the incorportion of [3H]-fucose and [3H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500-600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with 125I-labeled wheat germ agglutinin (WGA) and Maackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas 125I-peanut agglutinin bound only to the HMW glycoconjugates RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.

Topics: Agglutinins; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Electrophoresis, Polyacrylamide Gel; Galactose; Glucosamine; Glycoconjugates; Glycosylation; Head and Neck Neoplasms; Humans; Lectins; Leucine; Mannose; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Molecular Weight; Tretinoin; Tumor Cells, Cultured

Both retinoic acid (RA) and the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) have shown efficacy in head and neck cancer chemoprevention trials. To compare their activity and mechanism of action, the 1483 oral head and neck squamous cell carcinoma (HNSCC) cell line was grown in organotypic culture, an in vitro system that allows cellular stratification and simulates carcinoma in situ, and was exposed to 10 micromol/L of either RA or 4HPR. Extensive apoptosis, as evidenced by in situ deoxyribonucleic acid end-labeling, occurred in 4HPR-treated cultures after 9 days, with >80% cell loss (P< .001). In contrast, the growth of cultures treated with RA was inhibited by only 32%, with no evidence of apoptosis. Because 4HPR has low systemic toxicity and is a potent inducer of apoptosis in HNSCC cells, its role in chemoprevention of head and neck cancers, including cancers that are resistant to RA-induction therapy, warrants further investigation.

Topics: Apoptosis; Carcinoma, Squamous Cell; Drug Screening Assays, Antitumor; Fenretinide; Head and Neck Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

Retinoids have been shown to act as cytostatic agents against a variety of tumor cell types, including squamous carcinoma cells. Recently it was reported that certain retinoids can induce apoptosis as well. Because we are investigating the potential of retinoids in chemoprevention and therapy for head and neck premalignant and malignant lesions, we compared the effects of all-trans-retinoic acid (ATRA) and N-(4-hydroxyphenyl)retinamide (4HPR) on seven human head and neck squamous cell carcinoma cell lines (17A, 17B, 22A, 22B, 38, SqCC/Y1, and 1483). Six of the seven cell lines showed dramatic morphological changes after treatment with 10 micrometer 4HPR, whereas no such changes were induced by 10 micrometer ATRA. To determine whether these retinoids can induce apoptosis, we analyzed both detached and attached cells after 2, 5, and 7 days of treatment for evidence of DNA fragmentation by DNA electrophoresis on agarose gels. In five of the seven cell lines, a DNA ladder was observed after treatment with 10 micrometer 4HPR for 5 or 7 days, whereas treatment with ATRA resulted in a less pronounced effect. In 17B cells, a clear DNA ladder was observed as early as 2 days after treatment with 4HPR; however, neither ATRA nor 9-cis-retinoic acid was as effective. In addition, morphological changes associated with apoptotic cell death, such as chromatin condensation and nuclear segmentation, were observed by propidium iodide staining and by electron microscopic analysis after 4HPR treatment. These results demonstrate that 4HPR causes apoptosis in several head and neck squamous cell carcinoma cell lines and that it is more potent in this effect than ATRA.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Fenretinide; Head and Neck Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

We have previously shown that beta-all trans retinoic acid (RA) inhibits macrocellular growth of a multicellular tumor spheroid model for squamous carcinoma, as measured by spheroid size, but allows for continuing DNA synthesis and cell cycle progression, the two being reconciled by a cell death effect. DNA synthesis in the presence of growth inhibition suggested a rationale for examining combination chemotherapy with RA-inhibited cells. To this aim, we have extended this observation to a series of 8 squamous carcinoma cell lines. Cells were treated with 1 microM RA for 7 days and cell growth parameters monitored. Although growth inhibition ranged from 0% (A431) to approx. 80% (MDA 886Ln), [3H]-thymidine incorporation (cpm/microgram protein) and percent S-phase (by flow cytometry) in 7-day RA-treated cells was equal or higher than in their control vehicle-treated cells in 7/8 SCC cell lines. Thus RA-induced growth inhibition is not just cytostasis. Combination therapy was examined with MDA 886Ln, MDA 686Ln, 1483 and A431 cells pre-treated for 7 days with 1 microM RA followed by cisplatin or 5-fluorouracil treatment. An increased effectiveness for the combination was shown using 5-day tetrazolium dye (MTT) growth assays when cells were growth-inhibited by RA. Computerized analysis of data using median-effect and isobologram techniques indicated that the interaction of RA with these chemotherapeutic agents was synergistic. With squamous carcinoma, RA treatment inhibits growth while allowing for continuing DNA synthesis, and these RA-treated, growth-inhibited cells exhibit increased sensitivity to chemotherapeutic agents.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line; Cell Survival; Cisplatin; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Therapy, Combination; Fluorouracil; Head and Neck Neoplasms; Humans; S Phase; Tretinoin; Tumor Cells, Cultured

The ability of all-trans-retinoic acid (RA) to modulate the growth and squamous differentiation of four head and neck squamous cell carcinoma cell lines (183, 886, 1483, and SqCC/Y1) was examined, and the relationship of their state of squamous differentiation and RA responsiveness to the expression of cytosolic RA-binding proteins (CRABPs), nuclear RA receptors (RARs), and retinoid X receptors (RXRs) was investigated. RA inhibited proliferation of all but the 183 cell line and suppressed squamous differentiation markers K1 keratin, type 1 transglutaminase, and involucrin mRNAs and proteins to varying degrees in 183, 1483, and SqCC/Y1 cells. Traces of CRABP-I mRNA were detected only in the 886 cells, whereas CRABP-II mRNA was detected in the other three cell lines. RA suppressed CRABP-II expression in SqCC/Y1 cells but had no effect on its expression in the other cell lines. All cell lines expressed mRNAs for RAR-alpha, RAR-beta, RAR-gamma, and RXR-alpha. The RAR-beta mRNA level was lowest in the SqCC/Y1 cells, and RXR-beta and RXR-gamma were not detected in any of the cell lines. RA treatment increased the levels of the three RAR mRNAs in most of the cell lines but had no effect on the RXR mRNAs. The CRABP-II mRNA level in SqCC/Y1 cells was lowest in cells grown in serum-free medium and increased when the cells were grown in medium with 5 or 10% serum. In contrast, the RXR-alpha mRNA level was inversely related to serum concentration. The results show that, in head and neck squamous cell carcinoma cells, there are no simple relationships among the expression of CRABPs, RARs, and RXRs and either squamous differentiation or response to RA-induced growth inhibition or suppression of squamous differentiation.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Head and Neck Neoplasms; Humans; Keratins; Protein Precursors; Receptors, Retinoic Acid; RNA, Messenger; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Intrinsic fluorescence spectroscopy offers a new method for diagnosing head and neck cancers. By establishing a unique spectral fingerprint for benign tissue, one can readily identify subtle changes in tissue based on altered spectral patterns. The authors applied this technology to a multicellular tumor spheroid (MTS) model and obtained baseline spectral data. A cohort of MTS was treated with the chemopreventive agent retinoic acid (RA) to determine its effect on tumor cells. Excitation and emission spectroscopy were performed on the samples. Spectroscopic scans demonstrated consistently that RA-treated MTS exhibit a decrease in the peak associated with reduced nicotinamide-adenine dinucleotide (NADH) and an increase in the peaks associated with flavins, tryptophan, and cytokeratins when compared to controls. These findings are suggestive of alterations in cellular electron transport, an increase in proteins incorporating tryptophan, and a decrease in adenosine triphosphate (ATP) in the RA-treated cells. A discussion of the potential clinical applications of intrinsic fluorescence spectroscopy is included.

Topics: Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Spectrometry, X-Ray Emission; Tretinoin; Tumor Cells, Cultured

The protective effects of ascorbic acid (AA), n-acetyl-l-cysteine (NAC), alpha-tocopherol acid (ATA), alpha-tocopherol-acid succinate (TAS), and 13-cis-retinoic acid (CRA) on mutagen-induced chromosomal breakage were studied. Mutagen-sensitivity was determined by the bleomycin assay in human lymphoblastoid cell lines (LCLs) and cultures of peripheral blood lymphocytes (PBLs) from head and neck cancer patients. Preincubation with chemopreventive agents statistically significantly decreased mutagen-induced chromatid breakage in LCLs and PBLs in a dose-related manner. As the concentration of the agents was increased in tenfold increments in the study range, mean breakage rates were reduced by 3.0 to 7.7% in LCLs and by 6.0 to 11.1% in PBLs. The effective concentrations are comparable to those achieved in clinical applications and found in human dietary studies. A similar phenomenon in vivo, if identified, may explain the differences in occurrence of head and neck and other cancers between populations with different dietary habits. The bleomycin assay may be used for studying compounds with presumed chemopreventive properties.

Topics: Acetylcysteine; Anticarcinogenic Agents; Antimutagenic Agents; Ascorbic Acid; Bleomycin; Cell Line; Chromosome Aberrations; Head and Neck Neoplasms; Humans; Lymphocytes; Tretinoin; Vitamin E

The epidermal growth factor receptor (EGF-R) gene is overexpressed or amplified in various human squamous cell carcinomas, including those of the head and neck (HNSCC). Earlier we found that beta-all-trans-retinoic acid (RA) inhibited the growth and suppressed the aberrant squamous cell differentiation of several cultured HNSCC cell lines. Here we examined the effects of RA on the expression and function of EGF-R in two HNSCC cell lines, 1483 and 183, which exhibit distinct states of squamous cell differentiation, EGF-R mRNA levels, and responses to the growth inhibitory effects of RA. Treatment with RA (1 microM, 7 days) of the RA-sensitive 1483 cells decreased the level of EGF-R mRNA two- to four-fold and the binding of 125I-EGF to the cell surface by 30%-35%. In contrast, RA treatment of the 183 cells did not alter the EGF-R mRNA level or the binding of 125I-EGF. Other effects of RA on EGF-R structure and function were similar in both cell lines. RA did not alter the amount of immunoprecipitable [35S]methionine-labeled cellular EGF-R, 125I-cell surface labeled EGF-R, EGF-R internalization, or transforming growth factor alpha (TGF-alpha) mRNA. More important, RA treatment of both cell lines decreased EGF-R autophosphorylation activity detected in immune-complex-kinase assay by about three- and five-fold in the 1483 and 183 cells, respectively. Likewise, RA decreased the glycosylation of EGF-R in both cell lines. In the 1483 cells, RA suppressed the incorporation of either glucosamine or fucose by about 50%, whereas in the 183 cells RA suppressed the incorporation of fucose by about 80%. These results demonstrate that RA can modify the structure of the EGF-R by decreasing its glycosylation and suggest that these changes may suppress the autophosphorylation activity of the receptor kinase. The RA-induced changes in EGF-R do not correlate with the effect of RA on the growth of the cells but may be related to the suppression of squamous cell differentiation in the 1483 cells.

Topics: Carcinoma, Squamous Cell; Cell Membrane; ErbB Receptors; Glycosylation; Head and Neck Neoplasms; Humans; Phosphorylation; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Retinoids (vitamin A analogues) inhibit the squamous differentiation of normal and malignant epithelial cells. This study investigated the ability of the head-and-neck squamous-cell carcinoma (HNSCC) cell line 1483 to undergo squamous differentiation in the absence and presence of beta-all-trans retinoic acid (RA). The growth of these cells in culture is accompanied by an increase in keratinocyte transglutaminase, involucrin and keratin KI, 3 established markers of squamous cell differentiation. Higher levels of these differentiation markers were detected in cells cultured in delipidized serum (DLS), from which endogenous retinoids have been extracted, than in cells cultured in fetal bovine serum (FBS), which contains retinoids. Treatment with I microM RA decreased the levels of the various differentiation markers in cells cultured in either FBS or DLS as revealed by immunofluorescent labelling of permeabilized cells and by immunoblotting of cell extracts using specific monoclonal or polyclonal antibodies. The cells' ability to cross-link proteins to form envelopes under the plasma membrane was stimulated in the presence of calcium ionophore but inhibited by RA. These results indicate that the malignant 1,483 HNSCC cells recapitulate the main characteristics of normal squamous-cell differentiation in culture and that RA suppresses this differentiation as it does in normal keratinizing epithelial cells.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Immunoblotting; Ionophores; Keratinocytes; Keratins; Neoplasm Proteins; Protein Precursors; Retinoids; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cholesterol Esters; Depression, Chemical; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Sulfotransferases; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Line; Culture Techniques; Head and Neck Neoplasms; Humans; Retinoids; Tretinoin; Tumor Cells, Cultured

Cell line MDA 886Ln was established from a laryngeal lymph node metastasis. When grown as a multicellular tumor spheroid (MTS), it exhibits squamous differentiation. We studied the effects of beta-all-trans retinoic acid (RA) on the growth, differentiation and glycoprotein content of this MTS model for squamous carcinomas of the head and neck. The growth of MTSs was inhibited in a dose-dependent manner by 10(-6) to 10(-10) M RA. Growth inhibition occurred between 3 and 5 days of RA treatment (10(-6)M). Immunohistochemical and electrophoretic analyses revealed that RA suppressed the morphological markers of squamous differentiation (squames), involucrin expression, and keratin expression. Gly-coprotein expression was examined by metabolic labelling using 3H-glucosamine, in situ labelling of polyacrylamide gels with 125I-labelled wheat-germ agglutinin (WGA), localization of fluorescein isothionate-WGA in frozen sections, and determination of sialyltransferase activity. Treatment using 10(-6) M RA altered glycoprotein expression both biochemically and morphologically, and WGA was shown to bind preferentially to sialic acid residues. The sensitivity of this MTS model to RA treatment and its ability to be analyzed through morphological, immunohistochemical and biochemical techniques suggest that it will prove useful in studying the relationships between growth, differentiation and RA-induced alterations in squamous carcinomas.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Glycoproteins; Head and Neck Neoplasms; Humans; Keratins; Male; Molecular Weight; Neoplasm Proteins; Organoids; Protein Precursors; Tretinoin; Tumor Cells, Cultured

Evidence from several in vitro systems indicates that cellular responses to DNA topoisomerase II-reactive compounds (i.e., the epipodophyllotoxins and intercalating agents) may be affected by the relative rate of proliferation. Using a human head and neck squamous carcinoma cell line 183A, we have investigated the effect of beta-all-trans-retinoic acid (RA), a substance with known antiproliferative effects, on the DNA cleavage and cytotoxic activities of etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide which interact with topoisomerase II. The effect of RA treatment on the activity of X-radiation and bleomycin, both of which produce free radical mediated effects, was also examined. RA treatment (10 to 20 microM for 72 h) does not significantly influence DNA cleavage induced by X-radiation or bleomycin but decreases DNA cleavage and cytotoxicity mediated by etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide. Further, this effect can be demonstrated at a dose of RA that is minimally growth inhibitory. The inhibitory effect of RA appears to be localized to the nucleus given that similar effects on drug-mediated DNA cleavage can be demonstrated in nuclei isolated from RA-treated cells. However, both drug-stimulated DNA cleavage activity and topoisomerase II catalytic activity are approximately equal in crude nuclear extracts of untreated and RA-treated cells. These data suggest that the resistance to topoisomerase II-reactive drugs induced by RA treatment of 183A cells is not mediated through a direct effect on the enzyme, but, instead, is related to other changes in the nuclear milieu occurring in the initial stages of quiescence such as altered chromatin conformation.

Topics: Amsacrine; Carcinoma, Squamous Cell; Cell Survival; DNA Damage; DNA Topoisomerases, Type II; Etoposide; Head and Neck Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

The effects of butyrate and retinoic acid in combination with catecholamines or histamine on the HN-1 human head and neck squamous carcinoma cell line were investigated analysing cell proliferation, placental alkaline phosphatase (PLAP) activity, and relative cytokeratin content. Butyrate inhibited cell proliferation in agar, whereas retinoic acid induced a small inhibitory effect. Butyrate enhanced PLAP activity in a time related manner in contrast to retinoic acid, which had no significant effect. However, retinoic acid inhibited the efficacy of butyrate to induce PLAP activity. A synergistic enhancement of PLAP activity was demonstrated after treatment of butyrate pretreated cells with catecholamines or histamine. The beta-adrenergic antagonist propranolol partly inhibited the aforementioned enhancement of PLAP activity, whereas the alpha-adrenergic antagonist phentolamine further enhanced PLAP activity. Indirect labeling of keratins with a polyclonal antibody showed that cytokeratin content was enhanced by butyrate but not by retinoic acid. Further analysis of cytokeratin content using four monoclonal antibodies showed that labeling of cytokeratins (5 + 8) was increased by butyrate. PLAP activity could be modulated by a concerted action of either butyrate plus retinoic acid or butyrate plus catecholamines or histamine, indicating a possible role for PLAP in tumour cell proliferation.

Topics: Alkaline Phosphatase; Butyrates; Butyric Acid; Carcinoma, Squamous Cell; Catecholamines; Cell Division; Cell Line; DNA; Flow Cytometry; GPI-Linked Proteins; Head and Neck Neoplasms; Histamine; Humans; Isoenzymes; Keratins; Phentolamine; Placenta; Propranolol; Tretinoin

As a part of an assessment of the potential use of retinoids in preventive and adjuvant treatment of HNSCC, we examined the effects of beta-all-trans retinoic acid (RA) on the growth and cell-surface glycoconjugates of 2 HNSCC cell lines. These lines, designated 1483 and 183A, were established from an untreated patient with a well-differentiated SCC of the retromolar trigone and one with a poorly differentiated SCC of the tonsil. Whereas the 1483 cells were sensitive to RA in that their anchorage-dependent growth, their colony growth on solid substratum, and their anchorage-independent growth in semi-solid agarose gel were all inhibited in a dose-dependent fashion by RA concentrations in the range between 1 nM and 10 microM, the 183A cells were not inhibited by RA. Their anchorage-dependent growth and colony formation were stimulated by RA, whereas their anchorage-dependent colony formation was not altered. Cell-surface glycoconjugates were modulated by RA in the sensitive 1483 cells but not in the 183A cells. Treatment of the 1483 cells resulted in a large increase in the cell-surface labelling of high-molecular-weight (Mr greater than 400,000) galactoglycoconjugates and sialoglycoconjugates, as well as an Mr 280,000 sialoglycoconjugate. Glycoconjugates with similar electrophoretic mobilities in polyacrylamide gels were labelled intensely on the surface of the 183A cells even before RA treatment and only minor changes were noticed in their labelling after treatment. These results demonstrate that RA can exert different effects on different HNSCC lines, and suggest that correlations might exist between responsiveness to RA and the stage of differentiation of the HNSCC, and between modulation of cell growth and enhancement of cell-surface glycoconjugate glycosylation by RA.

Topics: Carbohydrates; Carcinoma, Squamous Cell; Cell Line; Galactosamine; Galactose; Glycosylation; Head and Neck Neoplasms; Humans; Molecular Weight; N-Acetylneuraminic Acid; Sialic Acids; Tretinoin

A pharmacokinetic study of 13-cis-retinoic acid was performed in nine patients following administration of a single oral dose of 80 mg. An average lag time of 1.2 hours was observed, followed by fast absorption, with a mean half-life of 0.5 hour. Peak plasmatic concentration of 733 ng/ml occurred at 2.3 hours. The disposition profile showed a rapid distribution half-life of 1.3 hours and a terminal elimination half-life of 24.7 hours. No 13-cis-retinoic acid was detected unchanged in urine. An important interpatient variability was noted.

Topics: Administration, Oral; Chromatography, High Pressure Liquid; Drug Evaluation; Half-Life; Head and Neck Neoplasms; Humans; Isotretinoin; Kinetics; Male; Tretinoin

The serum levels of retinol, RBP (retinol-binding protein) and PALB (prealbumin) were found to be significantly lower in patients with malignant tumors of the head and neck region than in controls. In tumor tissues as well as in normal laryngeal mucosa, specific binding sites for retinol and retinoic acid were found. Whereas retinol-binding (CRBP = cellular retinol-binding protein) could only be detected in a few cases, binding for retinoic acid (CRABP = cellular retinoic acid-binding protein) was present in all specimens investigated. The presence or lack of binding sites was not dependent on the actual serum retinol levels. With regard to the antineoplastic role of vitamin A, the reduced serum levels are considered as a possible factor in tumor development and growth. CRBP and CRABP are assumed to be mediating factors for the retinol and retinoic acid action. Since the presence of CRABP is a constant finding, we propose that retinoic acid and its synthetic derivatives with high affinity for CRABP could be appropriate antineoplastic drugs in these tissues.

Topics: Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Laryngeal Mucosa; Male; Middle Aged; Prealbumin; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Vitamin A

In some squamous cell carcinomas of the otorhinolaryngologic region with different grades of differentiation, a protein was found that specifically binds vitamin A acid. In 28 of 37 tumors, the retinoic acid-binding sites were found in significant amounts, according to the authors' data. Areas with metastases showed a lower incidence of retinoic acid-binding, whereas in all normal epiglottis and vocal cord tissue specimens the binding was present. The possible significance of the protein-binding for the biologic effect of the vitamin A acid is discussed.

Topics: Binding Sites; Binding, Competitive; Carcinoma, Squamous Cell; Centrifugation, Density Gradient; Etretinate; Head and Neck Neoplasms; Humans; Neoplasms; Otorhinolaryngologic Diseases; Protein Binding; Tretinoin; Vitamin A

A phase I study of 13-cis-retinoic acid was done in 16 patients with head and neck malignancies using a modified Fibonacci search scheme, with individual doses ranging from 20 to 120 mg/m2. Drug doses greater than 60 mg/m2 induced intense headaches, urethritis, desquamative dermatitis, vertigo, and ataxia. The severity of these side effects precludes the use of 13-cis-retinoic acid as a potential chemopreventive agent at doses greater than 60 mg/m2.

Topics: Adult; Aged; Cheilitis; Dose-Response Relationship, Drug; Drug Evaluation; Female; Head and Neck Neoplasms; Headache; Humans; Ichthyosis; Isomerism; Male; Middle Aged; Tretinoin; Urethritis