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Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of hydrogen peroxide (H(2)O(2)) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 degrees C because the H(2)O(2) concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H(2)O(2) produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H(2)O(2) because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H(2)O(2) decomposition. The effect of the glucose oxidation rate, i.e., the H(2)O(2) production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H(2)O(2) formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H(2)O(2) decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.

We evaluated the orally administered S-1, in combination with ionizing radiation both in vivo and in vitro against human oral cancer cell lines. Human oral cancer cell lines were used as subcutaneous xenografts in nude mice. S-1 (10 mg/kg) was administered orally 1 h before radiation treatments (1.5 Gy), or 1 h after radiation for five consecutive days. Apoptotic cells were detected by TUNEL method. For in vitro analysis, attached cells were treated with S-1 (50 microg/ml) for 1 h and then irradiated (3, 6, 9, 12, 15 Gy), or they were treated with S-1 for 1 h after radiation. Cell survival was determined by clonogenic assay. The combination of S-1 and radiation was more effective than either agent alone. In addition, S-1 administration before radiation was more effective than S-1 administration after radiation. Moreover, the combination of S-1 and radiation could induce apoptosis significantly than either agent alone (P < 0.01). In vitro clonogenic survival experiments demonstrated the dose enhancement ratio of 1.22 (radiation + S-1), 1.45 (S-1 + radiation) in B88 cells, and 1.16 (radiation + S-1), 1.28 (S-1 + radiation) in HSG cells. These data demonstrate that the combination of S-1 and fractionated radiotherapy is more effective against human oral cancer xenografts than either agent alone, and that S-1 administration before radiation is more effective than after radiation, suggesting a potential clinical applicability of combination treatment of S-1 and radiation in oral cancer therapies.

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-gamma and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4(-/-)) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4(-/-) mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4(-/-) mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

It is well known that loss of the cyclin-dependent kinase inhibitor p27Kip1 protein correlates with the poor prognosis of various cancers including oral squamous cell carcinoma (SCC). Posttranslational degradation of p27Kip1 protein is mediated by phosphorylation of Thr-187 of p27Kip1 protein, which follows ubiquitination. In this study, we constructed an expression vector expressing mutant type p27Kip1 gene (pcDNA3.1-p27Kip1 mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), which is not influenced by ubiquitin-mediated degradation. We transfected mutant and wild type p27Kip1 genes into an oral SCC cell line, B88 to up-regulate the expression of mutant or wild p27Kip1 gene in each transfectant. B88-p27Kip1 mt showed significant growth inhibition than B88-p27Kip1 wt or B88-neo in vitro (p < 0.01). Also, both types of B88-p27Kip1 showed G1 arrest and apoptosis, however, B88-p27Kip1 mt showed remarkable G1 arrest. In addition, B88-p27Kip1 mt and B88-p27Kip1 wt showed markedly inhibition of the migration and out-growth of cancer cells than B88-p27Kip1 wt or B88-neo. Moreover, B88-p27Kip1 mt also showed remarkable suppression of tumor growth and cervical lymph metastasis than B88-p27Kip1 wt or B88-neo in vivo (p < 0.01). In short, the mutant type p27Kip1 gene could show more potent antitumor effects than wild type p27Kip1 gene in B88 cells. These findings suggest that mutant type p27Kip1 gene has the potential to become a novel and powerful gene therapy tool for patients with oral cancers.

Background: The inducible nitric oxide synthase (iNOS) is associated with inflammatory processes and cancer formation through production of nitric oxide (NO). However, the clinical importance of the expression of iNOS in oral cancer remains unclear. In the present study, we examined whether up-regulation of the iNOS gene can affect growth and metastasis of an oral cancer cell line (B88t cell) in vitro and in vivo., Materials and Methods: We constructed an expression vector containing sense-oriented murine iNOS cDNA with pcDNA3.1. We transfected B88t cells with the sense expression vector to up-regulate the expression of the iNOS gene in the sense transfectants., Results: The expression of iNOS protein was up-regulated in the sense transfectants and that up-regulation of the iNOS gene exerted a growth inhibitory effect on B88t cells in vitro and in vivo. Moreover, up-regulation of the iNOS gene markedly inhibited the migration of cancer cells in a Boyden chamber. Furthermore, up-regulation of the iNOS gene dramatically inhibited metastases to the cervical lymph node in vivo., Conclusion: These findings suggest that up-regulation of the iNOS gene may suppress the tumorigenicity and metastasis of oral cancer cells.

We have demonstrated the possibility that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system might be involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC). In order to further clarify the role of the SDF-1/CXCR4 system in oral SCC, we examined the expression of CXCR4 and SDF-1 in biopsy specimens from 61 patients with oral SCC by means of immunohistochemistry, and investigated several clinicopathological factors, including age, sex, lymph node metastasis, invasion, recurrence and prognosis. The expression of CXCR4 and SDF-1 was observed in 57.3 and 11.4% of our subjects, respectively. Although we were unable to find a statistically significant association between the expression of SDF-1 and any clinicopathological factors, we did find a significant association between the expression of CXCR4 and lymph node metastasis (P=0.0417). Moreover, the mode of invasion (P=0.0002) and recurrence of the tumors (P=0.0185) were strongly associated with CXCR4 expression, and the CXCR4-positive group showed a significantly poorer prognosis than the CXCR4-negative group (P=0.0401). Recombinant SDF-1alpha stimulated in vitro invasiveness and scattering in CXCR4-expressing oral SCC cells, but the treatment did not affect the expression of matrix metalloproteinases or urokinase-type plasminogen activator. These results indicate that SDF-1/CXCR4 signaling in oral SCC cells might be involved in the diverse action of oral SCC, including invasion or micrometastasis at the primary site and lymph node metastasis.

We examined the mechanism involved in the induction of apoptosis in human oral cancer (B88) cells with 5-fluorouracil (5-FU) and cisplatin (CDDP) combination. Three different combination treatment sequences were evaluated: i) 5-FU administered simultaneously with CDDP for 72 h (sequence I); ii) CDDP administered for 24 h before 5-FU treatment for 48 h (sequence II); and iii) 5-FU administered for 24 h before CDDP treatment for 48 h (sequence III). When combining the two drugs at doses 40% of their respective cytotoxicity, the growth-suppressing ratios were 49, 55, and 40% in sequences I, II, and III, respectively. Caspase 3 was significantly activated in sequence II as compared to its level of activation in sequence I or III. The mitochondrial release of cytochrome c was much greater in sequence II than in sequence III. In addition, activation of caspase 8 was most strongly activated in sequence II as compared with sequences I and III. Activation of caspase-9 was also observed in sequence II, and, to a lesser extent, in sequence III. Finally, although Bcl-2 was reduced in sequence II, no significant change was observed in sequence III. Accordingly, the data presented here demonstrate that sequence II showing a significant increase in the induction of apoptosis of cancer cells, is superior to sequences I and III.

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-kappaB-dependent reporter plasmid, AILb-A induced NF-kappaB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-kappaB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to gamma-irradiation (IR) and 5-Fluorouracil (5-FU) in human oral carcinoma cells (B88) in which NF-kappaB activity was constitutively suppressed. Three super-repressor form of IkappaBalpha cDNA-transfected cell (B88mI) clones and 1 empty vector-transfected cell clone (B88neo) have been established. We found that the tumor-forming 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)-1alpha, 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-kappaB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes., (Copyright 2003 Wiley-Liss, Inc.)

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Recently, we have demonstrated that PPARgamma is expressed in human salivary gland tumors and its ligands inhibit the growth of cultured salivary gland cancer cells. However, expression and function of PPARgamma in normal and neoplastic human oral squamous epithelium remains unclear. In the present study, we examined PPARgamma expression in human oral squamous cell carcinoma (OSCC) and tested its ligands for any antitumor effect. PPARgamma mRNA was detected by RT-PCR in some OSCC tissues and cultured cells, but the PPARgamma protein showed neither expression nor ligand-induced transcriptional activity. Despite loss of PPARgamma function, synthetic PPARgamma ligands caused significant dose-dependent inhibition of cancer cell growth. These results suggest that PPARgamma function is inactivated in OSCC cells and the anti-proliferative effect of its synthetic ligands is independent of PPARgamma.

Tuberous sclerosis is caused by mutations in tuberous sclerosis complex (TSC) 2 on chromosome 16p13.3, encoding tuberin which is thought to be essential for p27(Kip1) to regulate the cell cycle. In this study, we conducted to examine whether overexpression of TSC2 can affect the growth of oral cancer cells which have different expression level of p27(Kip1) protein. We constructed an expression vector containing sense-oriented rat TSC2 cDNA with pcDNA3.1. We transfected oral cancer cells, B88t (high expression of p27(Kip1) protein) and HI (low expression of p27(Kip1) protein) with the sense expression vector to up-regulate the expression of TSC2 gene. Overexpression of TSC2 exerted the growth inhibitory effect of B88t and HI in vitro and in vivo. These findings suggest that overexpression of TSC2 may exert the antitumor effect on oral cancer cells whether they have high expression of p27(Kip1) protein or not.

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.

p27Kip1 is a cyclin-dependent kinase inhibitor which regulates progression of cells from G1 into S phase in a cell cycle. p27Kip1 has been reported to be an important prognostic factor in various malignancies. As the ability of invasion and metastasis has been thought to be the most important factor for patient's prognosis, we examined whether up-regulation or down-regulation of p27Kip1 can affect the ability of invasion and metastasis of an oral cancer cell (B88t cell) in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human p27Kip1 cDNA with pcDNA3.1. We transfected B88t cells with the empty vector, the sense- or antisense-oriented expression vector to regulate the expression of p27Kip1 gene. Up-regulation of p27Kip1 protein markedly inhibited the migration of cancer cells in a Boyden chamber. Down-regulation of p27Kip1 protein markedly enhanced the migration of cancer cells in a Boyden chamber, and the tumor invasion in nude mice. Moreover, we detected up-regulation of E-cadherin and down-regulation of Tiam-1 (tumor invasive and metastasis-1) in the sense transfectants, up-regulation of Tiam-1 and down-regulation of E-cadherin in the antisense transfectants by Western blotting. These results suggest that p27Kip1 may play an important role in the invasion and metastasis of an oral cancer cell involved in regulation of E-cadherin and Tiam-1.

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.

Saliva contains a number of proteins and glycoproteins that protect oral tissues, but little is known about the role of human saliva in innate immunity. Here we showed that human major salivary gland cells constitutively expressed a bacterial pattern recognition receptor, CD14, by immunohistochemistry. Human salivary gland cells in culture express CD14 mRNA and a 55-kDa CD14 protein in, but not on the cells, and secrete a soluble form with the same molecular mass. Human whole saliva contains a 55-kDa CD14, and the concentration of parotid saliva was 10-fold higher than whole saliva, which is comparable to that of serum CD14. Levels of CD14 in unstimulated whole and parotid saliva were unchanged before and after a meal and between unstimulated and stimulated saliva, indicating that saliva CD14 is constitutively secreted into the oral cavity. In contrast, lipopolysaccharide (LPS)-binding protein was below the detectable level. The saliva CD14 is functionally active in that it mediated the activation of CD14-lacking intestinal epithelial cells by LPS in a Toll-like receptor 4-dependent manner. These results suggested that saliva CD14 is important for the maintenance of oral health and possibly intestinal homeostasis.

Cepharanthine is a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata. The drug has been widely used for the treatment of many acute and chronic diseases and can exert antitumor effects on several human cancer cells. In this study, we examined the mechanism of the antitumor effects by cepharanthine on a human oral squamous cell carcinoma (SCC) cell line. Treatment of oral SCC cells with cepharanthine (10-20 micrograms/ml) resulted in a significant suppression of cell growth. Cepharanthine preferentially suppressed the growth of B88 cells when compared with other human oral SCC cells. Moreover, it was found, by flow cytometry analysis and Hoechst 33258 staining, that G1 arrest and DNA fragmentation occurred in cepharanthine-treated B88 cells. Furthermore, induction of p27Kip1 and reduction of cyclin E and Skp2 were detected by Western blotting. B88 tumor-bearing nude mice were treated with cepharanthine, which was administered subcutaneously (40 mg/kg/day). The cepharanthine treatment results in a significant suppression of tumor growth. Overall these results indicate that cepharanthine may inhibit the growth of human oral SCC cells by down-regulating cyclin E to induce G1 arrest through a pathway of p27Kip1 induction.

Background: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity., Methods: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided., Results: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups., Conclusions: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

It is important to augment the anti-cancer host response in cancer treatment. Recent studies suggested that the signaling via Toll-like receptors (TLRs) which are newly identified receptor molecules recognizing many pathogens, are involved in the induction of anti-cancer immunity. Seya et al. demonstrated that maturation of dendritic cells (DCs) and cytokine induction by the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) are induced via both TLR2 and TLR4. Akira et al. discovered a new molecule of TLR family, TLR9, recognizing unmethylated bacterial CpG-DNA, whose clinical use is expected for cancer therapy as a potent inducer of a helper T cell 1 (Th1)-type T-cell response. TLR9-deficient mice did not show any responses to CpG-DNA, including Th 1 cytokine production and maturation of DCs. We have obtained two molecules, a lipoteichoic acid-related molecule isolated from streptococcal agent OK-432, and a plant-derived 55-kDa protein that can induce Th1 response and elicit a strong anti-cancer effect in vivo and in vitro. Our basic experiments demonstrate that TLR4 signaling is intimately involved in anti-cancer immunity induced by these immunopotentiators. Our clinical examination in oral cancer patients also suggests the requirement of both TLR4 and MD-2 in the OK-432-induced anti-cancer host response. Establishment and clinical use of the methodology for human cancer therapy by utilizing TLR signaling is greatly expected.

Background: OK-PSA, a lipoteichoic acid (LTA)-related molecule isolated from a streptococcal agent OK-432, enhances anti-tumor immunity as a potent inducer of Th1-type cytokines. Recently, we obtained the data suggesting that natural killer (NK) cells may play a significant role for OK-PSA-induced cytokine production in vitro., Materials and Methods: We conducted the animal experiments using athymic nude mice bearing human salivary adenocarcinoma to examine the role of NK cells in OK-PSA-induced anti-tumor immunity. OK-PSA was peritumorally injected into the mice. Cytokines in the sera were analyzed by ELISA. mRNAs for cytokines were detected by RT-PCR. 51Cr release test was performed to measure killer cell activities., Results: OK-PSA markedly increased the amounts of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18 that are generally called "Th1-type cytokines" in the sera derived from tumor-bearing nude mice, and also accelerated the killing activities of tumor-infiltrating lymphocytes as well as of draining lymph node cells. Furthermore, OK-PSA administration resulted in significant inhibition of tumor growth, but the effect of OK-PSA was almost completely inhibited by the deletion of NK cells using anti-asialo GM1 antibody., Conclusion: These findings strongly suggested that NK cells are closely involved in OK-PSA-mediated anti-tumor immunity.

p27(Kip1) is a cyclin-dependent kinase inhibitor which regulates progression of cells from G1 into S phase in a cell cycle. Loss of p27(Kip1) has been associated with disease progression and an unfavorable outcome in several malignancies. In the present study, we conducted to examine whether up-regulation or down-regulation of p27(KiP1) can affect the growth of oral cancer cell (B88 cell) in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human p27(Kip1) cDNA with pcDNA3.1(Invitrogen). We transfected B88 cells with the sense or antisense expression vector to regulate the expression of p27(Kip1) gene in each transfectant. The expression of p27(Kip1) protein was up-regulated in the sense transfectants and down-regulated in the antisense transfectants. Moreover, up-regulation of p27(Kip1) protein exerted the growth inhibitory effect, and down-regulation of p27(Kip1) protein enhanced the growth of B88 cells in vitro and in vivo. Furthermore, we detected the G1 arrest and sub-G1 peak in the sense transfectants by flow cytometry analysis. These results suggest that up-regulation of p27(Kip1) protein may exert the growth inhibitory effects through induction of G1 arrest and apoptosis on oral cancer cell line.

Background: p27Kip1 is a cyclin-dependent kinase inhibitor which regulates the progression of cells from the G1-into the S-phase in a cell cycle. Loss of p27Kip1 is associated with disease progression and an unfavorable outcome in several malignancies. The purpose of this study was to determine whether p27Kip1 expression can be a useful prognostic factor in oral squamous cell carcinoma (SCC) patients., Patients and Methods: p27Kip1 expression was investigated by immunohistochemistry in tissue samples from 81 patients with oral SCC. The associations between p27Kip1 expression and clinicopathological characteristics and patient survival were also analyzed., Results: Significant associations were found between p27Kip1 expression and histological grade (p = 0.010), therapeutic effect (p = 0.004) and patient outcome (p = 0.005). The 5-year survival rates of p27Kip1 high- and low-expression tumours were 80.4% and 56.7%, respectively, this difference being significant (p = 0.009) by log-rank test. Multivariate analysis revealed that reduced term survival was related to low levels of p27Kip1 expression (p = 0.008) and advanced stage (stages III and IV) (p = 0.003)., Conclusion: These results suggest that reduction of p27Kip1 plays an important role in the progression of oral SCC and is considered to be a useful prognostic factor in oral SCC patients.

It is important to examine the abnormality of the entire p53 tumor suppressor pathway in head and neck cancer. We examined the mRNA expressions of p53 regulatory factors, p33ING1 and p14ARF, and a p53-target gene, p21WAF1 in head and neck cancer. Nine of 14 benign pleomorphic adenomas (PAs) and 7 of 8 malignant salivary gland tumors (MSGTs) expressed p33ING1 mRNA. Thirteen of 14 PAs expressed p14ARF mRNA, however, only 1 of 8 MSGTs expressed p14ARF mRNA. Eight of 14 PAs and 7 of 8 MSGTs expressed p21WAF1 mRNA. In salivary gland tumors, there was clear correlation between the expression of p33ING1 and p21WAF1 (p<0.0001, r2=0.53). However, there was no correlation between the expression of p14ARF and p21WAF1 (p=0.6543, r2=0.009). Twenty-six of 28 oral squamous cell carcinomas (SCCs) expressed p33ING1 mRNA. Nineteen of 28 oral SCCs expressed p14ARF mRNA. All of the oral SCCs expressed p21WAF1 mRNA. In oral SCCs, the expressions of both p33ING1 (p=0.009, r2=0.181) and p14ARF (p=0.0009, r2=0.271) correlated with the expression of p21WAF1. Interestingly, 24 of 26 oral SCCs (92%) showed either abnormality of p53 itself or loss of expression of p53 regulatory factors, p33ING or p14ARF. These results suggest that head and neck cancer often involve the dysfunction of p53 tumor suppressor pathway.

Objective: Our previous results suggested that suppression of tumor necrosis factor alpha (TNFalpha)-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 TNFalpha-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 TNFalpha alone or with a combination of TNFalpha 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 TNFalpha was examined., Results: Although TNFalpha 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 TNFalpha and cepharanthine. In addition, cepharanthine suppressed the TNFalpha-stimulated NF-kappaB activity by partly preventing the degradation of IkappaBalpha protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha 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.

The transcription factor NF-kappa B inhibits the apoptotic response induced by TNF-alpha. However, in salivary gland cell clones (ACMT-6 and ACMT-7) in which NF-kappa B activation was suppressed by introduction of a super-repressor form of I kappa B-alpha cDNA, TNF-alpha did not cause apoptosis. Thus, to investigate the molecular mechanism involved in the unresponsiveness of these cell clones to TNF-alpha-induced apoptosis, we examined the effect of TNF-alpha on the expression of antiapoptotic proteins, including TNF receptor-associated factor (TRAF)-1, TRAF-2, cellular inhibitor of apoptosis protein (cIAP)-1, and cIAP-2. Here we show that expression of TRAF-1 was commonly detected by treatment with TNF-alpha in ACMT-6, ACMT-7, and an empty vector-transfected cell clone (ACpRc-1) and that downregulation of TRAF-1 protein by either treatment with an antisense oligonucleotide or introduction of an antisense plasmid resulted in the induction of apoptosis in these cell clones. Our results, therefore, suggest that one of the mechanisms by which cells acquire resistance to TNF-alpha-induced apoptosis is a TNF-alpha induction of TRAF-1.

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage., ((c)2002 Elsevier Science (USA).)

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. It is expressed in several tissue types, including adipose tissue in which it stimulates adipogenesis. Recent studies have demonstrated that PPARgamma ligands induce cellular differentiation and inhibit cell growth in carcinomas of various organs including the breast, prostate, lung, colon, stomach, bladder, and pancreas. However, whether PPARgamma is expressed in human salivary gland tumors and its function in this tissue is unknown. In the present study, we examined PPARgamma gene expression in human salivary gland cancer cells and tested its ligands for any antitumor effect. PPARgamma mRNA was detected by RT-PCR in both benign and malignant salivary gland tumor tissues. The effect of PPARgamma on cell growth was investigated using four human salivary gland cancer cell lines; HSG, AZA3, HSY and TYS, which were confirmed to express PPARgamma1 mRNA and protein. Retinoid X receptor alpha protein, which forms heterodimers with PPARgamma, was also detected in all the cells tested. Data obtained by luciferase assay indicated that the intrinsic PPARgamma protein was activated by the synthetic ligands, troglitazone and pioglitazone, but not by the natural ligand, 15-deoxy-delta12, 14-prostaglandin J2. The synthetic PPARgamma ligands, particularly troglitazone, caused significant dose-dependent inhibition of cancer cell growth. Furthermore, the overexpression of PPARgamma1 or PPARgamma2 in cancer cells also reduced significantly their growth rate. These results suggest that PPARgamma and its synthetic ligands suppress the growth of human salivary gland cancer cells and it may be a useful molecular target for cancer treatment.

Several investigators have demonstrated that TGF-beta stimulated clone-22 (TSC-22) regulates cell growth and differentiation, and cell death. TSC-22 is a putative transcriptional regulator containing a leucine zipper-like structure and a nuclear export signal. We previously showed the cytoplasmic localization of TSC-22 and the nuclear translocation of TSC-22 concomitant with induction of apoptosis in salivary gland cancer cells. In the present study, we attempted to identify the active domain of TSC-22 protein that regulated the biological functions of TSC-22. We constructed three mammalian expression vectors, which could produce full length TSC-22 only in cytoplasm, the leucine zipper structure of TSC-22 in both cytoplasm and nucleus, and the leucine zipper structure only in nucleus. Then, we transfected a salivary gland cancer cell line, HSG with these expression vectors, and investigated the growth profile of the transfectants. None of the TSC-22 constructs inhibited the monolayer growth and the anchorage-dependent colony formation of HSG cells. However, the leucine zipper structure of TSC-22 markedly inhibited the anchorage-independent colony formation of HSG cells (p<0.001; one way ANOVA). Full length TSC-22 also suppressed the anchorage-independent colony formation of HSG cells, although the effect of full length TSC-22 was much lower than those of the leucine zipper constructs. These observations suggest that the leucine zipper structure in TSC-22 protein is an active domain that negatively regulates the growth of salivary gland cancer cells.