Factors affecting the growth of head and neck squamous carcinoma cell lines

Cell plating density influences the growth pattern of human squamous cell carcinoma (SCC) cell lines in culture. SCC lines were used to study factors responsible for these effects. Experiments in which the medium was changed daily or in which conditioned medium was used showed that the growth-supporting factors were not found in the media. However, when cells were plated in dishes in which the same cell line had been grown to confluence and removed by scraping, logarithmic growth began immediately. The effect was not seen on areas of the plate that had been covered with coverslips or in dishes where fibroblasts had been cultured. Antibodies to fibronectin, laminin, and a variety of epidermal cell antigens were used to determine the nature of the growth-stimulating substances remaining on the dish following cell removal. Results indicate that an extracellular matrix with similarities to basement membranes was present on the conditioned dishes.     

Effects of a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on growth, apoptosis, gene expression, and chemosensitivity in head and neck squamous cell carcinoma cell lines

BACKGROUND: Recent studies provide evidence that the constitutive activation of nuclear factor-kappa B, NF-kappaB plays a critical role in enhancing the growth of several types of malignancies, including head and neck squamous cell carcinoma (HNSCC). METHODS: In this study, we examined the effects of a newly synthesized NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on growth, induction of apoptosis, gene expression, and chemosensitivity in two HNSCC cell lines (YCU-H891 and KB), which expressed high levels of nuclear NF-kappaB protein. RESULTS: DHMEQ showed strong growth inhibitory effects on these two cell lines, with a 50% cell growth inhibition (IC50) concentration of approximately 20 microg/mL. These growth inhibitory effects were associated with inhibition of the NF-kappaB activity. Treatment with DHMEQ induced apoptosis in a dose-dependent manner accounting, at least in part, for the growth inhibition by DHMEQ. DHMEQ strongly inhibited cyclin D1 and vascular endothelial growth factor (VEGF) promoter activity and decreased the levels of cyclin D1 protein and VEGF mRNA in KB cells. In addition, low concentrations of DHMEQ (1.0 or 5.0 microg/mL) synergistically enhanced the cellular sensitivity of YCU-H and KB cells to cisplatin, which is a key chemotherapeutic agent in the treatment of HNSCC. CONCLUSIONS: These results suggest that DHMEQ may be effective when used alone or in combination with other agents in the treatment of HNSCC.     

Effects of glutamine on head and neck squamous cell carcinoma.

Glutamine supplements are being studied as protectants against the mucositis associated with radiation therapy of head and neck squamous cell carcinoma. Glutamine is an important amino acid for the maintenance of healthy gut function and nitrogen transport. It is also a preferred energy source for many types of tumors through an altered Krebs cycle. The effects of glutamine supplements on the growth of head and neck squamous cell carcinoma have not been studied. Eighteen nude mice were implanted with one established cell line, and 18 were implanted with another. The mice were paired and fed either a glutamine-rich or a glutamine-poor diet. There was not a statistically significant difference in the growth rates of the xenografts or in the weight gain of the mice between the diet groups. These findings are encouraging for the continued use of glutamine supplements in patients with head and neck squamous cell carcinoma.     

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

Transforming growth factor-β is known to be a potent autocrine growth inhibitor produced by a wide variety of cells, including cells of the immune system. Other investigators have noted that the growth of nontransformed keratinocytes is inhibited by transforming growth factor-β, whereas various carcinoma cell lines are resistant to these effects. Head and neck squamous cell carcinoma cells are known to have surface receptors for this cytokine. We thus assessed the effect of transforming growth factor-β on the growth of head and neck squamous cell carcinoma cell lines. Four head and neck squamous cell carcinoma cell lines were incubated with varying concentrations of transforming growth factor-β, and cytotoxicity was evaluated with a methylene blue colorimetric assay. After culturing in transforming growth factor-β for 4 days, inhibition of growth was detected in CAL-27 (maximal inhibition at 5.0 ng/ml), UMSCC-1, and UMSCC-19 (maximal inhibition at 50 ng/ml) cell lines. One other cell line, UMSCC-8 was found resistant to the inhibitory effects of transforming growth factor-β. Kinetics analysis experiments revealed minimal inhibition before day 2 of incubation, at which time inhibition increased linearly to day 4. Assessment of double-stranded DNA fragmentation suggested that DNA fragmentation occurs before significant cytotoxicity. Electron microscopic analysis and gel electrophoresis of extracted DNA revealed morphologic features consistent with apoptotic cell death. Our findings indicate that transforming growth factor-β significantly inhibits the growth of head and neck squamous cell carcinoma cell lines by inducing apoptotic cell death. (OTOLARYNGOL HEAD NECK SURG 1995;112:728-34.)     

Photodynamic effects of anthracyclin derivatives on squamous cell carcinoma cell lines of the head and neck

BACKGROUND AND OBJECTIVE: In the last years, photodynamic therapy, performed with hematoporphyrin derivatives, gained in importance for the treatment of superficially situated malignomas. The use of hematoporphyrin as photosensitizer is limited especially by the low depth of penetration and its side effects. The aim of the present study was to evaluate the effectiveness of photodynamic therapy with anthracyclin derivates in squamous cell carcinoma cell lines. STUDY DESIGN/MATERIALS AND METHODS: The photodynamic effects of the anthracyclin derivates adriamycin and epirubicin as well as the effects of the hematoporphyrin derivatives photofrin-II and photosan-3 were examined and compared in 10 squamous cell carcinoma cell lines derived from head and neck tumors. RESULTS: Beside their cytostatic effect, the applied cytostatics revealed a marked photodynamic effect. A statistically significant difference for photodynamic effects of both cytostatic agents and the hematoporphyrin derivates could not be shown. CONCLUSIONS: These results revealed that the above mentioned cytostatics could be considered as possible alternative photosensitizer for photodynamic therapy.     

Effects of IFI16 overexpression on the growth and doxorubicin sensitivity of head and neck squamous cell carcinoma-derived cell lines

BACKGROUND: In a previous analysis of head and neck squamous cell carcinomas (HNSCCs), we showed that the levels of the interferon-inducible protein IFI16 inversely correlate with cancer grade. In this study, we further evaluate the molecular role of IFI16 in the development of HNSCCs. METHODS: The effect of IFI16 expression was evaluated by its retroviral restoration in an IFI16-negative HNSCC-derived cell line, HNO136. Growth rate and soft agar colony formation were evaluated. The effect of IFI16 restoration in cells exposed to doxorubicin was also analyzed. RESULTS: IFI16 restoration resulted in the inhibition of both cell growth and in vitro transforming activity and increased doxorubicin-induced cell death by accumulating the cells at the G2/M phase. CONCLUSION: In agreement with our previous in vivo data, IFI16 appears to be involved in maintaining the normal growth of epithelial cells, whereas its downregulation may contribute to uncontrolled cell proliferation and tumorigenesis.     

Effects of 5-azacytidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines.

OBJECTIVE: To determine the cellular effects of 5-azacytidine (5-azaC) and sodium butyrate on two human liver cancers, HepG2 and Hep3B. SUMMARY BACKGROUND DATA: Primary liver cancer is a significant health problem; treatment options are limited and prognosis is poor. Recent studies have focused on the role that programmed cell death (i.e., apoptosis) plays in both normal and neoplastic growth: certain genes can either suppress (e.g., Bcl-2, Bcl-xL) or promote (e.g., Bik, Bax, Bak) apoptosis. The identification of novel agents targeted to specific molecular pathways may be beneficial in the treatment of this disease. METHODS: Human liver cancer cell lines HepG2 and Hep3B were treated with 5-azaC alone, butyrate alone, or 5-azaC and butyrate. Morphologic and proliferative changes were assessed by light microscopy and 5-bromo-2'-deoxyuridine staining; flow cytometry was used to determine cell cycle characteristics. Apoptosis was assessed by DNA laddering and the in situ apoptosis detection assay using the TdT-mediated dUTP nick end labeling method. In addition, total RNA and protein were analyzed by ribonuclease protection and Western blot, respectively, to assess changes in the expression of apoptosis-related genes. RESULTS: Treatment with either 5-azaC or butyrate inhibited cell growth and induced apoptosis in both HepG2 and Hep3B cells; the combination of 5-azaC and butyrate was not more effective than either agent alone. 5-azaC alone resulted in a more differentiated-appearing morphology and G2 cell cycle arrest in both cell lines. Treatment with 5-azaC or butyrate affected the expression levels of proteins of the Bcl-2 family. CONCLUSIONS: Both 5-azaC and butyrate induced apoptosis in the HepG2 and Hep3B liver cancer cells; 5-azaC treatment alone produced G2 arrest in both cell lines. Proteins of the Bcl-2 family may play a role in the cellular changes that occur with treatment, but further studies are required to define this potential role. Products of the apoptotic pathway may prove to be useful therapeutic targets in the treatment of hepatic cancers.     

Non-steroidal anti-inflammatory drugs inhibit telomerase activity in head and neck squamous carcinoma cell lines.

BACKGROUND: Antiproliferative effects in neoplastic cells of different origin have been attributed to non-steroidal anti-inflammatory Drugs (NSAIDs) during the past few decades. METHODS: We tested the influence of NSAIDs and hydrocortisone on cell lines derived from head and neck squamous cell cancer (HNSCC) and on normal oral mucosal keratinocytes. Cell numbers were assayed by cell counting, proliferation, telomerase activity with a colorimetric assay, and cell cycle distribution by flow cytometry. RESULTS: In the neoplastic cell lines indomethacin and ibuprofen caused a dose-dependent reduction of cell numbers and telomerase activity without altering cell viability and increased the percentage of cells in G0/G1 phase. In normal oral mucosal keratinocytes, only minor effects could be detected in response to NSAIDs and hydrocortisone. CONCLUSION: These results demonstrate that NSAIDs have activity against HNSCC cells in vitro and may have clinical applications in combination with other therapeutic regimens.     

P53 mutation correlates with cisplatin sensitivity in head and neck squamous cell carcinoma lines

BACKGROUND: A critical factor for successful organ preservation treatment in head and neck cancer may be selecting tumors that respond to chemotherapy and radiation. Previous results in patients indicated that tumors that overexpressed p53 were more sensitive to chemotherapy than those that did not overexpress p53. METHODS: To determine the relationship of p53 mutations to sensitivity to cisplatin in vitro, 23 head and neck squamous cell carcinoma (HNSCC) cell lines were analyzed for cisplatin sensitivity, p53 expression, and p53 mutation status. RESULTS: Mutations of the p53 gene were identified in 13 of 23 of the cell lines tested. Mutation of the p53 gene was significantly associated with high levels of expression of the p53 protein. The average ID(50) (drug dose required to inhibit 50% of cell growth) for cell lines with mutant p53 was 6.8 microM, whereas the average ID(50) for cell lines with wild-type p53 was 13.7 microM. CONCLUSIONS: These in vitro data support a role for mutation of the p53 tumor suppressor gene as a marker for response to cisplatin in HNSCC.     

Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisense cyclin D1

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of ³ H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with nontransfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells. (Otolaryngol Head Neck Surg 1998;119:593-9.)     

Squamous cell carcinoma radioimmunoassay in squamous cell carcinoma of the head and neck

Tumor-associated antigen has shown promise as a clinical aid in the detection and monitoring of uterine cervical squamous cell carcinoma. Antigen levels have been shown to reflect the extent of disease and response to treatment. These findings have suggested that measurements of tumor-associated antigen may be useful in monitoring other squamous cell carcinomas. To test this hypothesis, we measured tumor-associated antigen using the squamous cell carcinoma radioimmunoassay in 103 patients with previously treated squamous cell head and neck tumors and 28 patients with known squamous cell carcinoma of the head and neck. Increased squamous cell carcinoma antigen levels were found in 39 percent of patients with known tumors and in 19 percent of the patients with previous curative resection. The sensitivity of the assay limited its usefulness in predicting the presence of new and recurrent tumors.     

Correlation of biomarkers in head and neck squamous cell carcinoma

Objective

To evaluate the relationship of functional magnetic resonance imaging (MRI) parameters, including choline/creatine ratio (Cho/Cr) and apparent diffusion coefficient (ADC) with protein expression of 10 common tumor and prognostic markers in head and neck squamous cell carcinoma.

Study Design

Cross-sectional study.

Setting

University hospital.

Subjects and Methods

The Cho/Cr and ADC obtained from 74 patients with head and neck squamous cell carcinoma were correlated with the expression level of the 10 protein markers as determined by immunohistochemistry.

Results

Cho/Cr showed significant positive correlations with cyclooxygenase 2 in primary tumors (r = 0.714), and epidermal growth factor receptor in metastatic cervical lymph nodes (r = 0.522). ADC showed significant (r = −0.591) negative correlation with human epidermal growth factor receptor 2 in metastatic cervical lymph nodes.

Conclusion

There are relationships between protein and functional MRI markers. Future research in this direction may improve our understanding of the cancer micro-environment.     

Inhibitory effect of p27KIP1 gene transfer on head and neck squamous cell carcinoma cell lines

BACKGROUND: Although p27 gene mutations are rarely found in cancer, the level of p27 protein expression decreases during tumor development. In several tumors, including laryngeal cancer, decreased expression of p27 is associated with a poor prognosis. METHODS: The proliferation-inhibitory effect of p27 gene transfer on human head and neck squamous cell carcinoma (HNSCC) cell lines (SNU-1041, SNU-1066, and SNU-1076) by adenoviral vector was investigated. RESULTS: On transduction of the human HNSCC cell line with adenovirus-p27 (Ad-p27), a high level of p27 expression and increase of cyclin D1 and E were observed. Cell cycle analysis showed a marked decrease in the proportion of cells in S phase and an increase in G1 phase. Soft agar clonogenic assay showed a marked decrease in clonogenicity. CONCLUSION: These results suggested that overexpression of p27 could show proliferation-inhibitory effects on HNSCC cell lines. Thus, gene therapy using adenovirus-p27 seemed to have a potential to develop into a new cancer gene therapy modality.