Yoichi Yamada

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes,HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novoformation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K m values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 × 105 to 1 × 106 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 × 105 to ∼2 × 106 Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 × 105 to ∼2 × 106 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 × 106 Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

In Vitro Synthesis of Hyaluronan by a Single Protein Derived from Mouse HAS1 Gene and Characterization of Amino Acid Residues Essential for the Activity

HAS1 was expressed as a FLAG-tagged HAS1 fusion protein in COS-1 cells. This recombinant protein was extracted with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) from the membrane fraction and purified by anti-FLAG affinity chromatography and subsequent SDS-polyacrylamide gel electrophoresis. A protein solubilized from the one single band on the gel was able to synthesize hyaluronan when incubated with UDP-GlcNAc and UDP-GlcA as donor substrates without any further additions. The detergent-solubilized and purified HAS1 protein, however, exhibited quite different kinetic properties from the membrane-bound protein. When assayed under the reconstitutive conditions where the reaction mixture was layered onto the buffer containing high concentration of CHAPS, the activity was enhanced and the kinetic properties became similar to those of the membrane-bound protein. In addition, a HAS1 gene product by an in vitro transcription/translation system also showed HAS1 activity under the reconstitutive conditions. To our surprise, when incubated with UDP-GlcNAc alone, the protein was found to synthesize chito-oligosaccharide. Taking advantage of these enzyme reaction properties, active sites on the protein involved in for hyaluronan and chito-oligosaccharide synthesis were characterized. Site-directed mutagenesis induced in the cytoplasmic central loop domain of the protein revealed that several amino acid residues conserved among those domains of various proteins of a HAS family were essential for both hyaluronan and chito-oligosaccharide syntheses but one of them was not for chito-oligosaccharide synthesis. The substitutions that caused partial or severe loss of the activity gave no significant changes of the K m values of the mutated proteins, suggesting that no conformational or other indirect changes were involved in the effect. Taken together, the results suggest that the HAS1 protein alone is able to synthesize hyaluronan and different amino acid residues on the cytoplasmic central loop domain are involved in transferring GlcNAc and GlcA residues, respectively.

Elevated transcript level of hyaluronan synthase1 gene correlates with poor prognosis of human colon cancer

Hyaluronan plays important roles in the complex processes of tumor invasion and metastasis. It is now known that three hyaluronan synthase (HAS) isoforms catalyze hyaluronan synthesis, which raises the question of how they are involved in malignant tumor progression. In this study, we examined the correlation between tumor progression and transcriptional levels of three HAS isoforms in specimens of human colon cancers. Tumor tissues from 31 patients with different diagnostic grades were assessed to determine the level of each HAS isoform by real time RT-PCR. The mean expression coefficients for HAS1, HAS2 and HAS3 in the cancerous parts were 0.82-, 0.91- and 1.22-fold, respectively; of those in the noncancerous parts at Dukes stage A; 1.00-, 0.95- and 1.06-fold, respectively, at stage B; and 1.95-, 1.16- and 1.19-fold, respectively, at stage C. In survival analysis, a significant correlation was observed between poor survival and the HAS1 transcript level. When the ratio of tumor to normal tissue in the HAS1 level was compared with that of the HA receptor transcript level, there was a positive correlation with that of the CD44 variant 6 level at Dukes stage C. Our current results therefore suggest that HAS1 plays a role in the malignant progression of human colon cancer cells.

Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells

BackgroundOral squamous cell carcinoma (OSCC) accounts for the majority of oral cancers. Despite recent advances in OSCC diagnostics and therapeutics, the overall survival rate still remains low. Here, we assessed the efficacy of a combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment in human OSCC cells.MethodsThe combinatorial effect of ATO/CDDP on the growth and apoptosis of OSCC cell lines HSC-2, HSC-3, and HSC-4 was evaluated using MTT and annexin V assays, respectively. Chou–Talalay analyses were preformed to evaluate the combinatorial effects of ATO/CDDP on the dose-reduction index (DRI). To clarify the mechanism underlying the ATO/CDDP anticancer effect, we also examined the involvement of reactive oxygen species (ROS) in ATO/CDDP-induced apoptosis.ResultsCombination index (CI) analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in HSC-2 cells, with CI values ranging from 0.78 to 0.90, where CIu2009<u20091 defines synergism. The CI values in HSC-3 and HSC-4 cells ranged from 0.34 to 0.45 and from 0.60 to 0.92, respectively. In addition, ATO/CDDP yielded favorable DRI values ranging from 1.6-fold to 7.71-fold dose reduction. Compared to mono-therapy, ATO/CDDP combinatorial therapy significantly augmented the loss of mitochondrial potential, caspase-3/7 activity and subsequent apoptosis. These changes were all abrogated by the antioxidant N-acetylcysteine.ConclusionsThis study provides the first evidence for a synergistic ATO/CDDP anticancer (apoptotic) activity in OSCC cells with a favorable DRI, thereby highlighting its potential as a combinational therapeutic regime in OSCC.

Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines

OBJECTIVESnPlumbagin (PL), a naturally occurring quinoid, exerts antitumoral effects in diverse types of cancer cells. However, the effect of PL on tumor cell proliferation in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, we assessed the efficacy of PL, in human OSCC cells.nnnMETHODSnThe effect of PL on the cell growth and apoptosis of OSCC cell lines was evaluated using MTT and Annexin V assays, respectively. The effect of PL on mitochondrial membrane potential loss and reactive oxygen species (ROS) generation was evaluated using flow cytometry analysis.nnnRESULTSnMTT assay showed that PL dose-dependently suppressed OSCC cell growth, with IC50 values ranging from 3.87 to 14.6 μM. Flow cytometry analysis revealed that PL treatment resulted in a significant decrease in mitochondrial membrane potential and an increase in the number of apoptotic cells. Notably, ROS generation was significantly elevated after PL treatment. Furthermore, a ROS scavenger, N-acetylcysteine (NAC), clearly suppressed the decrease in mitochondrial membrane potential, increase of caspase-3/7 activity, and apoptosis after PL treatment.nnnCONCLUSIONnThis study provides the considerable evidence of the tumor-suppressive effect of PL, thereby highlighting its therapeutic potential for OSCC treatment.

Activated cytotoxic T-lymphocyte immunotherapy is effective for advanced oral and maxillofacial cancers.

Conventional cancer treatments are surgery, radiotherapy, and chemotherapy, but treatment efficiency is insufficient and cancer recurrence is common. Immunotherapy has been added as an important cancer treatment component, but no reports on its efficacy in oral and maxillofacial cancers exist. We evaluated the clinical efficacy of adoptive immunotherapy using ex vivo-activated cytotoxic T lymphocytes (CTL) in the treatment of 7 patients with advanced oral and maxillofacial cancers with stage IV disease at diagnosis. The mean follow-up period was 26.2 months. Phenotype of the lymphocyte assay revealed that the percentage of CD4(+) T cells decreased and that of CD8(+) T cells increased among infused lymphocytes compared to that in unstimulated peripheral blood mononuclear cells (PBMCs), and infused lymphocytes produced a significantly higher level of IFN-γ than PBMCs or tumor cells alone. In a representative patient who refused surgery tumor regression was confirmed after CTL infusion. Computed tomography clearly indicated a significant reduction in tumor size followed by the complete disappearance of the tumor. Histological examination showed that the cancers in patients receiving CTL therapy were heavily infiltrated with lymphocytes. The other 2 patients who received CTL therapy as adjuvant therapy showed neither recurrent disease nor new disease lesions. The 1-year survival rates showing response and those with progressive disease were 100 and 25%, respectively. Moreover, no significant adverse reactions were reported during the study period. CTL therapy remains in the early stages of treatment options, but it has potential as a valuable treatment and improvement of quality of life for patients with otherwise incurable cancers.

Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines

NADPH oxidases, also known as the Nox family, are major sources of reactive oxygen species generation that regulate redox-sensitive signaling pathways. Recent studies have implicated the Nox family in cancer development and progression. However, the involvement of its members in the development of oral squamous cell carcinoma (OSCC) remains to be elucidated. To clarify this issue, we first analyzed mRNA expression of Nox/Duox family members (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) in five OSCC cell lines. Nox1 and Nox4 mRNAs were highly expressed in four OSCC cell lines. Western blot analysis revealed that the protein expression level of Nox1 was higher than that of Nox4 in the OSCC cell lines. In addition, knockdown of Nox1, but not Nox4, significantly suppressed cell viability and induced apoptosis in the HSC-2 and HSC-3 cells. We also found that a specific AKT inhibitor, perifosine, dose-dependently suppressed OSCC cell growth. Notably, Nox1 knockdown significantly attenuated the phosphorylation level of AKT. Furthermore, both Nox1 knockdown and perifosine treatment markedly enhanced the cisplatin-induced cytotoxic effect. Taken together, our results highlight that the Nox1/AKT signaling pathway plays an important role in cell survival in OSCC cells.