Koji Harada

Possible contribution of active MMP2 to lymph‐node metastasis and secreted cathepsin L to bone invasion of newly established human oral‐squamous‐cancer cell lines

We have established human oral‐squamous‐cancer cell lines, BHY and HN, derived from non‐metastatic cancer and metastatic cancer respectively. We examined the expression of matrix‐degrading enzymes and their inhibitors in these cell lines. Both cell lines expressed pro‐matrix metalloproteinase (MMP)1, proMMP2, proMMP9, membrane‐type MMP and urokinase‐type plasminogen activator. In addition to these enzymes, BHY cells secreted proMMP7 and procathepsin L, while HN cells secreted a large amount of active MMP2. BHY cells secreted a tissue inhibitor of matrix metalloproteinase, TIMP2, but only a trace level of TIMP1. Contrary to BHY cells, HN cells secreted TIMP1, but only a trace level of TIMP2. When we inoculated these cells into the masseter muscle of nude mice, both types of cell formed solid tumors, whose microscopic appearance was identical to that of the original tumors. BHY tumors were highly differentiated squamous‐cell carcinomas, and invasive to the masseter muscle and the mandibular bone. Despite their local aggressiveness, BHY tumors did not metastasize to any distant organs. HN tumors were poorly differentiated squamous‐cell carcinomas, weakly invasive to the muscle, but not to the mandibular bone. However, HN tumors frequently metastasized to cervical lymph nodes. These results suggest that the net activity of MMP2 (active MMP2/TIMP2) and cathepsin L secreted from cancer cells may contribute respectively to lymph‐node metastasis and to bone invasion by oral cancer cells.

An overview of the cell cycle arrest protein, p21WAF1

p21, also known as WAF1, Cip1, Sdi1, Mda 6 and Cap20 is a cell cycle protein that regulates and can arrest the cell cycle in G1 or S phase (either dependent or independent of p53). Its role may be pivotal in many cell processes including differentiation and apoptosis. This brief overview provides a summary of its presently known functions and indicates areas for further research, particularly in relation to oral malignant disease. Greater understanding of its role may lead to therapeutic advances in the management of malignant disease.

Enhanced radiosensitization and chemosensitization in NF-κB-suppressed human oral cancer cells via the inhibition of γ-irradiation- and 5-FU-induced production of IL-6 and IL-8

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to γ‐irradiation (IR) and 5‐Fluorouracil (5‐FU) in human oral carcinoma cells (B88) in which NF‐κB activity was constitutively suppressed. Three super‐repressor form of IκBα 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)‐1α, 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‐κB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

Therapy for oral squamous cell carcinoma by tegafur and streptococcal agent OK-432 in combination with radiotherapy: Association of the therapeutic effect with differentiation and apoptosis in the cancer cells

Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expres-sion of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p< 0.05) and DNA fragmentation rate ( p< 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p< 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.

Overexpression of p27Kip1 induces growth arrest and apoptosis in an oral cancer cell line

Abstract 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.

Cisplatin induces apoptosis in oral squamous carcinoma cells by the mitochondria-mediated but not the NF-κB-suppressed pathway

Cisplatin (CDDP) is a potent DNA-damaging anticancer agent, and its cytotoxic action is exerted by the induction of apoptosis. However, activation of the transcription factor NF-kappaB results in protection against apoptosis. We examined the molecular mechanisms involved in the induction of apoptosis by CDDP as regards both suppression of NF-kappaB and activation of caspases. Human oral squamous carcinoma cells (B88) were employed in this study. We found that CDDP treatment affected neither NF-kappaB activity nor the expression levels of antiapoptotic proteins, including TRAF-1, TRAF-2, and cFLIP, in B88 cells. However, two apoptosome molecules, cytochrome c and Apaf-1, were significantly augmented in the cytoplasm by CDDP treatment. Further, the activation of caspase-9 and caspase-3, downstream molecules leading to mitochondria-mediated apoptosis, were detected after treatment with CDDP. Finally, apoptosis was also clearly observed, as evidenced by cleavage of PARP through the activation of caspase-3. These findings suggest that CDDP exerts its apoptotic action by the mitochondria-mediated activation of caspases but not by the activation of caspases due to the inhibition of NF-kappaB activity that follows the suppression of antiapoptotic proteins.

Evidence of a bi-phasic effect of thrombospondin-1 on angiogenesis.

Contradictory results have been reported regarding the association between vascularity (used as an index of angiogenesis) and thrombospondin-1 (TSP-1) in human tumours. In previous studies, the reported association was based on the estimated average TSP-1 value per tumour, with a sufficient number of specimens collectively analysed per tumour type. Given the extent of intra-tumour heterogeneity, we determined the association between TSP-1 and vascularity within individual specimens, based on the average values of TSP-1 and vascularity in 10–20 pre-selected areas per tumour. Cells expressing TSP-1 mRNA were visualised by in situ hybridisation and quantified by point counting. Vascularity was quantified by point counting and vessel density of von Willebrand Factor-positive vessels. In 10 ductal breast carcinomas, a direct correlation between TSP-1 and vascularity was found in 4 tumours, no correlation in 3 and an inverse correlation in 3. The effect of TSP-1 on endothelial cell migration in vitro was assessed in the Boyden chamber assay. TSP-1 stimulated cell migration at low concentrations (0.1–10u2009μg/ml) and was inhibitory at high concentrations (25–100u2009μg/ml). These results suggest that TSP-1 may elicit a concentration-dependent, bi-phasic, effect on angiogenesis.

Characteristics of antitumour activity of cepharanthin against a human adenosquamous cell carcinoma cell line.

Cepharanthin is one of the biscoclaurine alkaloids widely used for treatment of many acute and chronic diseases; snakebite, bronchial asthma, alopecia areata, leukopenia during radiation therapy or anticancer treatment. Recently, it has been reported that cepharanthin exerts antitumour effects by increasing immunological competence of the host or apoptosis-inducing activity. In this study, we examined the antitumour effects of cepharanthin against a human adenosquamous cell carcinoma cell line (TYS). Treatment of TYS cells with cepharanthin (10 approximately 20 microg/ml) resulted in a significant suppression of cell growth. Moreover, it was found by the flow cytometry analysis, nick end labelling or agarose gel electrophoresis, that G1 arrest and DNA fragmentation occurred in cepharanthin-treated cells. In addition, it was detected that induction of p21(WAF1) protein and activation of caspase 3 protype, which is one of Interleukin-1beta converting enzyme (ICE) family proteases, were detected by Western blotting. The TYS tumour-bearing nude mice were treated with cepharanthin, which was administered subcutaneously (20 mg/kg/day). The cepharanthin treatment results in a significant suppression of tumour growth and an induction of apoptosis. These findings suggest that cepharanthin induces G1 arrest via expression of p21(WAF1) and apoptosis through caspase 3.

Expression of Vascular Endothelial Growth Factor in Normal and Tumour Oral Tissues Assessed with Different Antibodies

Expression of vascular endothelial growth factor (VEGF) in oral tissues was assessed using different antibodies. Quantitative and topographical differences were observed between paraffin and cryostat sections. Two polyclonal antibodies (PC36, PC37) differing in their cross-reactivity with VEGF121 (not recognized by PC36), were used to stain serial cryostat sections of normal oral mucosa (n=8) and squamous cell carcinoma (n=7). The expression of VEGF in the epithelium was overall higher with PC37 than with PC36, the difference being significant in normal oral mucosa (p=0.001) but not in squamous cell carcinoma samples (p=0.094). With PC36, VEGF expression was significantly higher in squamous cell carcinoma than in normal oral mucosa specimens, whereas the opposite was true with PC37. Our results suggest that the relative levels of isoform 121 to that of 165 (and possibly others) may be different in the tissues examined, with VEGF121 preferentially expressed in normal oral mucosa. Previously published conflicting results may, therefore, be due to the presence of variable ratios of VEGF isoforms in the tissues examined, combined with differences in the cross-reactivity of the antibodies used. VEGF isoforms 121, 165 and (for the first time) 189 were detected in two frozen oral tissues by polymerase chain reaction amplification. Quantification of specific VEGF isoforms will be required in future studies concerned with the clinical value of VEGF expression.

Characteristics of antitumor activity of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]- 2(1H)-quinolinone (vesnarinone) against a human adenoid squamous carcinoma-forming cell line grown in athymic nude mice.

The tumors produced by transplantation into nude mice of human adenoid squamous carcinoma-forming cell line TYS, presumably derived from a minor salivary gland, were treated with a differentiation-inducing agent, vesnarinone, which was given per o.s. daily at a dose of 200 mg/kg for 35 days. They were then examined morphologically and immunohistochemically. The vesnarinone treatment resulted in a significant suppression of tumor growth. In addition, tumor nests indicating keratinocyte and acinar cell differentiation were often observed in the treated tumors, but not in untreated controls. Tissue sections from vesnarinone-treated and untreated TYS tumors were stained with monoclonal antibody (NAb) directed to carbohydrate antigen LeY or proliferating cell nuclear antigen (PCNA) and with rabbit polyclonal antibody to p53. Antibody staining patterns were compared with morphological characteristics of cells as revealed by hematoxylin and eosin staining, and DNA fragmentation patterns as revealed by 3-OH nick-end labelling techniques. Tissue sections from vesnarinone-treated TYS tumors showed positive reaction with nick-end labelling and were extensively stained strongly by anti-LeY MAb, whereas the untreated tumors showed negative reaction with nick-end labelling and were infrequently stained by anti-LeY MAb. Within LeY-positive areas of tissue sections from the vesnarinone-treated tumors, keratinocyte and acinar cell differentiation as well as DNA fragmentation were frequently observed, although not all LeY-positive cells showed such signs of apoptosis. LeY-positive cells showed consistent negative staining by anti-PCNA MAb and anti-p53 rabbit serum. From these findings, it can be considered that vesnarinone has differentiation and apoptosis-inducing activity against TYS cells grown in athymic nude mouse.