We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to ␥-irradiation (IR) and 5-Fluorouracil (5-FU) in human oral carcinoma cells (B88) in which NF-B activity was constitutively suppressed. Three super-repressor form of I B␣ cDNA-transfected cell (B88mI) clones and 1 empty vector-transfected cell clone (B88neo) have been established. We found that the tumorforming ability in nude mice of B88mI clones was significantly lower than that of B88 or B88neo. This suppressed ability in tumorigenicity was attributed to the down-regulation of the expression of interleukin (IL)-1␣, IL-6, IL-8, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 in B88mI cell clones as compared to that in B88 or B88neo. IR and 5-FU induced a much greater degree of apoptosis, as evidenced by flow cytometry analysis and annexin V staining, in B88mI cell clones than in B88 or B88neo. When tumor-bearing nude mice were treated with IR or 5-FU, the suppression of tumor growth was significantly augmented in B88mI cell clones as compared to that in B88 or B88neo. ELISA analysis indicated that although a remarkable increase in production of IL-6 and IL-8 was observed in B88 and B88neo after in vitro exposure to IR or treatment with 5-FU, radiotherapy and chemotherapy-induced production of these cytokines was significantly suppressed in B88mI cell clones. These findings suggest that production of angiogenic factors and growth factors in response to radiotherapy and chemotherapy is a principal mechanism of inducible radioresistance and chemoresistance in human oral cancers, and establish the inhibition of NF-B as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

No abstract

Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

Objective. Our previous results suggested that suppression of tumor necrosis factor ␣ (TNF␣)-induced matrix metalloproteinase 9 (MMP-9) could prevent the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS). The present study was undertaken to investigate the effect of cepharanthine on the suppression of TNF␣-induced MMP-9 production in NS-SV-AC, an SV40-immortalized normal human acinar cell clone.Methods. After pretreatment with or without cepharanthine, NS-SV-AC cells were treated with TNF␣ alone or with a combination of TNF␣ and cepharanthine. The expression of MMP-9 was then examined at the protein and messenger RNA levels. In addition, the effect of cepharanthine on the morphogenetic behavior of NS-SV-AC cells cultured on type IV collagen-coated dishes in the presence of TNF␣ was examined.Results. Although TNF␣ induced the production of MMP-9 in NS-SV-AC cells, this production was greatly suppressed when cells were pretreated with cepharanthine, followed by treatment with both TNF␣ and cepharanthine. In addition, cepharanthine suppressed the TNF␣-stimulated NF-B activity by partly preventing the degradation of IB␣ protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen-coated dishes in the presence of both TNF␣ and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, pretreatment with cepharanthine restored the aberrant in vitro morphogenesis of the NS-SV-AC cells.Conclusion. These results may indicate a molecular mechanism by which cepharanthine is able to protect against the destruction of the acinar structure in salivary glands from patients with SS.

We have already demonstrated that human head and neck cancer cells have significantly enhanced levels of transcription factor nuclear factor (NF)-κB activity compared to their normal counterparts, suggesting that NF-κB plays an important role in the development of head and neck cancer. However, it has been reported that chemotherapeutic agents and radiation activate NF-κB activity in cancer cells, thus making the cells radioresistant and chemoresistant. In addition, we have shown that the suppression of NF-κB activity enhanced apoptosis in oral squamous cell carcinoma cells. In this study, we examined whether cepharanthin-induced inhibition of NF-κB activity enhances radiosensitivity in human oral carcinoma cells. Cepharanthin is a biscoclaurine alkaloid extracted from the roots of Stephania cepharantha hayata, and is widely used in Japan for the treatment of patients with leucopenia, nasal allergy, and venomous snakebites. γirradiation (IR) induces NF-κB activity in oral carcinoma cells through the activation of upstream molecules, including Akt and IκB kinase. However, a luciferase assay revealed that cepharanthin suppresses IR-induced NF-κB activity in oral squamous cell carcinoma cells, thereby enhancing the radio-sensitivity. Western blot analysis showed an enhanced cleavage of poly-(ADP-ribose) polymerase protein in carcinoma cells by both cepharanthin treatment and IR exposure compared to IR or cepharanthin alone. In an in vivo study, B88 cells were s.c. inoculated into the backs of nude mice. Tumor-bearing nude mice received either cepharanthin, IR alone, or a combination of cepharanthin and IR. The combined treatment suppressed tumor growth significantly more than either cepharanthin or IR alone. Cepharanthin inhibited the production of IR-induced IL-6 and IL-8, which are downstream targets of NF-κB. In quantitative real-time RT-PCR, IR also induced the expression of anti-apoptotic proteins [cellular inhibitor of apoptosis protein (cIAP)-1 and-2] in carcinoma cells. Treatment of cancer cells with cepharanthin combined with exposure to IR decreased cIAP-1 and-2 mRNA expression. These findings suggested that the combination of radiotherapy and cepharanthin could enhance radiosensitivity in the treatment of human oral cancer.