Longipetalol A: A Highly Modified Triterpenoid from Dichapetalum longipetalum.

Longipetalol A (1) is an unprecedented highly modified triterpenoid with a unique 1,2-seco-3-(2-oxo-phenylethyl)-17α-13,30-cyclodammarane skeleton, featuring an acetal-lactone fragment. It was isolated from Dichapetalum longipetalum along with two additional derivatives, namely, longipetalols B (2) and C (3). Their structures were elucidated using spectroscopic analyses combined with single-crystal X-ray diffraction. Compounds 1, 2, and 3 exhibited inhibitory effects on nitric oxide production in lipopolysaccharide-induced RAW264.7 macrophages.

Two new eunicellin diterpenoids from the East China Sea gorgonian Muricella sp.

A phytochemical investigation on gorgonian Muricella sp. from East China Sea resulted in the isolation of eight eunicellin diterpenoids including two new ones, muricellins A-B (1, 2). Chemical structures of these compounds were elucidated by spectroscopic techniques (1D and 2D NMR and MS) and by comparison with data reported in the literature. Anti-rheumatoid arthritis activities of 1, 3, 4, and 6 have been evaluated.

Three new polyunsaturated lipids from a Guangxi marine sponge Haliclona sp.

Three new polyunsaturated lipids, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraen-3-one (1), (6Z,9Z,12Z,15Z)-1-bromooctadeca-6,9,12,15-tetraen-3-one (2), and (Z)-ethyl docos-5-enoate (3), together with two known polyunsaturated lipids, 4(Z),7(Z),10(Z)-tridecatrienoic acid (4) and (6Z,9Z,12Z,15Z)-octadeca-1,6,9,12,15-pentaen-3-one (5), were isolated from the marine sponge Haliclona sp., which was collected from Guangxi, using HSCCC and HPLC methods. Chemical structures of the five compounds were elucidated by spectroscopic techniques.

Jaspiferins C-F, four new isomalabaricane-type triterpenoids from the South China Sea sponge Jaspisstellifera.

A chemical investigation of marine sponge Jaspis stellifera, collected from the South China Sea, led to the isolation of four new isomalabaricane-type triterpenoids, jaspiferins C-F (1-4). The structures of those compounds were elucidated by extensive spectroscopic methods. Jaspiferin C (1), which has the six-membered carbon ring at the side chain, was discovered for the first time from the isomalabaricane-type triterpenoids. The hypothesis of a biogenetic pathway to generate jaspiferin C (1) was depicted.

Dichapetalin-type triterpenoids from Dichapetalum longipetalum and their anti-inflammatory activity.

A phytochemical research on the twigs of Dichapetalum longipetalum (Turcz.) Engl. Resulted in five undescribed dichapetalin-type triterpenoids 1-5. Their chemical structures were determined by spectroscopic analysis of HR-ESIMS and NMR spectra and the absolute configuration of compound 1 was completely elucidated by single crystal X-ray crystallography. Through preliminary anti-inflammatory activity assessment, compound 1 exhibited inhibitory effect on LPS-induced NO production in RAW264.7 murine macrophages with an IC50 value of 2.09 µM.

Microarray gene analysis of Toll-like receptor signaling elements in chronic rhinosinusitis with nasal polyps.

OBJECTIVE: To identify the regulatory mechanisms of Toll-like receptor (TLR)-associated genes in chronic rhinosinusitis (CRS) with nasal polyps (NP) using gene microarray analyses. METHODS: We pooled: (1) NP biopsy specimens from 10 nonatopic CRS patients and (2) healthy mucosal tissue from 10 additional nonatopic healthy patients (controls). These pooled samples were evaluated by gene microarrays that included 125 genes for TLRs and associated signaling elements. To validate gene product expressions, 20 NP and 15 normal nasal turbinate tissues were evaluated for TLR-9 expression by immunohistochemical staining and Western blots using samples from gland cells, epithelial cells, and mononuclear cells cytologically identified by HE staining. RESULTS: In pooled NP samples compared to pooled controls, 4 genes were upregulated (≥ 2-fold higher expression) and 19 were downregulated (≤ 0.5-fold lower expression). TLR-9 was an upregulated gene in NP tissue. Compared to control tissue, there were significantly higher percentages of TLR-9 positively stained NP gland cells, epithelial cells, and mononuclear cells (p < 0.001). On Western blots, while both normal and NP tissues expressed TLR-9 protein, the expression was significantly more pronounced for NP tissue (p < 0.001). CONCLUSIONS: Inflammation associated with CRS may be due to dysregulated innate immune elements, particularly TLR-9 and its associated signal transduction elements, which may impact upon prolonged activation of adaptive immune responses in the sinonasal mucosa.

Role of the alpha v-integrin subunit in cell proliferation, apoptosis and tumor metastasis of laryngeal and hypopharyngeal squamous cell carcinomas: a clinical and in vitro investigation.

The objective of this study was to investigate the role of alpha v-integrin subunit (ITGAV, CD51) in invasion and metastasis of the laryngeal and hypopharyngeal squamous cell carcinomas and to evaluate whether an antisense oligonucleotide sequence (ASONs) targeting ITGAV gene can result in proliferative inhibition and induce to apoptosis of laryngeal carcinoma cell lines (Hep-2). Firstly, a tissue microarray contained 75 primary carcinomas, 29 non-cancerous normal tissues and 20 metastatic lymph nodes was constructed and used to detect the expression of ITGAV by immunohistochemistry. The changes of ITGAV expression from each group were assessed and correlated to the clinical parameters of the patients. Secondly, the ASONs targeting ITGAV gene was transfected into Hep-2 cells in vitro. The proliferative ability of the cells after transfection was measured by MTT assay and the apoptosis was detected using flow cytometry. Results showed that the expression of ITGAV was significantly correlated with differentiation and lymph node metastasis of these cancers. In vitro test showed that the proliferative ability of Hep-2 cells was significantly inhibited by ASONs in a way of concentration- and time-depending mode, and a significant apoptosis of Hep-2 cells was also observed after ASONs transfection. In conclusion, the expression of ITGAV was significantly correlated with differentiation and metastasis of the laryngeal and hypopharyngeal carcinomas; down-regulation of ITGAV gene could inhibit proliferation of Hep-2 cells and induce to its apoptosis. These results suggest that ITGAV gene may become a promising prognostic marker and new treatment target for these cancers.

Cyr61/CCN1 overexpression induces epithelial-mesenchymal transition leading to laryngeal tumor invasion and metastasis and poor prognosis.

BACKGROUND: To examine the expression of cysteine-rich 61 (Cyr61/CCN1) protein in laryngeal squamous- cell carcinoma (LSCC) tissues, and its relationship with the tumor epithelial-mesenchymal transition (EMT), invasion, metastasis, and prognosis. MATERIALS AND METHODS: Immunohistochemistry was used to detect the expressions of Cyr61, Vimentin (Vim), and E-cadherin (E-cad) in 88 cases of LSCC tissues and 30 cases of tumor-adjacent normal tissues. Vim and E-cad were used as mesenchymal and epithelial markers, respectively, to determine the relationship between Cyr61 expression and the EMT of LSCC cells. In addition, clinical and histopathological data were combined to analyze the relationship between the positive-expression rates of Cyr61, Vim and E-cad and LSCC invasion, metastasis and prognosis. RESULTS: In LSCC tissues, Vim expression rate was significantly higher than that of the tumor-adjacent tissues, whereas E-cad expression rate was significantly lower than that of the tumor-adjacent tissues. The Vim expression rate was significantly higher in stages T3 and T4 than in stages T1 and T2 LSCC tissues, whereas E-cad expression rate was significantly lower in stages T3 and T4 than in stages T1 and T2 LSCC tissues. Compared to the group without lymph node metastasis, the Vim expression rate was significantly higher and the E-cad expression rate was significantly lower in the group with lymph node metastasis. The expression rate of Cyr61 was significantly higher in LSCC tissues than in the tumor-adjacent normal tissues. In addition, the Cyr61 expression rate was higher in stages T3 and T4 than in stages T1 and T2 LSCC, and higher in the group with lymph node metastasis than in the group without lymph node metastasis. The Vim expression rate was significantly higher in the Cyr61 positive group than in the Cyr61 negative group, whereas the E-cad expression rate was significantly higher in the Cyr61 negative group than in the Cyr61 positive group. Survival analysis indicated that survival rates of Cyr61 positive, Vim positive and E-cad negative groups were significantly lower than that of Cyr61 negative, Vim negative and E-cad positive groups, respectively. CONCLUSIONS: Cyr61 expression is closely associated with LSCC invasion and lymph node metastasis. Overexpression of Cyr61 may induce EMT and therefore leads to LSCC invasion and metastasis and poor prognosis. Cyr61 may become a new maker for clinical prediction of LSCC invasion and metastasis and a new target for LSCC treatment.

KAI1/CD82 and MRP1/CD9 serve as markers of infiltration, metastasis, and prognosis in laryngeal squamous cell carcinomas.

OBJECTIVE: The current study explored the expression of KAI1/CD82 and MRP1/CD9 and its significance in laryngeal squamous cell carcinoma (LSCC). METHODS: The expression levels of KAI1/CD82 and MRP1/CD9 in 100 LSCC tissue specimens, as well as in 30 para-LSCC non-carcinomatous tissue specimens randomly taken from the patients, were assessed using the quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry and correlations with pathological parameters of LSCC and their influence on survival function were analyzed. RESULTS: KAI1/CD82 and MRP1/CD9 showed basically consistent changes in both mRNA and protein expression. Their expression in the 30 LSCC specimens was significantly lower compared with that in the corresponding non-carcinous tissues (P < 0.01 or 0.05), notably correlating with TNM stage, differentiation degree, clinical stage, and lymphatic metastasis (P < 0.01 or 0.05), but not gender, age, and LSCC growth sites (P > 0.05). The median survival of patients with positive KAI1/CD82 and MRP1/CD9 protein expression was longer than that of patients with negative protein expression (P < 0.01 or 0.05). KAI1/CD82 protein expression negatively correlated with MRP1/CD9 protein expression in LSCC (χ(2) = 31.25, P < 0.01). CONCLUSION: KAI1/CD82 and MRP1/CD9 may jointly participate in the development of LSCC. They may serve as the markers for judging the infiltration, metastasis, and prognosis of LSCC.

A role for the parafascicular thalamic nucleus in the development of morphine dependence and withdrawal.

The parafascicular thalamic nucleus (nPf) is a critical relay in the ascending system that mediates motor control in the central nervous system (CNS). Yet, little is known about whether or not the nPf is involved in the development of morphine dependence and withdrawal. In the present study, kainic acid was used to chemically destroy the nPf in Wistar rats, and morphine dependence and withdrawal models were established. Morphine withdrawal symptoms score was evaluated in each group. An electrophysiological method was used to measure the changes in spontaneous discharge of nPf neurons. mu-Opioid receptor (MOR) mRNA level in nPf was detected using semi-quantitative RT-PCR. The ultrastructural alterations were examined by transmission electron microscopy. Results showed that the bilateral lesion of nPf had a marked influence on the development of morphine dependence and withdrawal. In order to address the mechanisms underlying, we found: (1) the average frequency and sum of nPf neurons that exhibited spontaneous discharge were increased in the morphine withdrawal group in comparison with the sham model group (P<0.05); (2) MOR mRNA level in the nPf of the morphine dependence group was decreased in comparison with that of the sham model group (1.45+/-0.38 vs. 5.37+/-0.94, P<0.01). In the morphine withdrawal group, which underwent 40 h withdrawal, the MOR mRNA level was higher than that in the morphine dependence group (2.97+/-0.73 vs. 1.45+/-0.38, P<0.05) but still lower than that in the sham model group (P<0.05); (3) the ultrastructural injuries of nPf neurons, which were in the nucleus, organelles and neuropil, were marked in the morphine dependent and withdrawal groups. Our study indicated that nPf played an important role in the development of morphine dependence and withdrawal. The results suggest that nPf may become a therapeutic target for treating morphine withdrawal syndrome.

[Expression of alpha-v integrin subunit in the laryngeal and hypopharyngeal squamous cell carcinomas and its relationship with tumor angiogenesis].

OBJECTIVE: To investigate the expression of integrin alpha-v subunit in laryngeal and hypopharyngeal squamous cell carcinomas (LHSCC) and to correlate the expression ratio with clinic and pathologic features of LHSCC, meanwhile, to investigate the relationship between the expression of integrin alpha-v subunit and tumor angiogenesis. METHODS: A tissue microarray of LHSCC was designed and made. Using this microarray, the expression of integrin alpha-v subunit in LHSCC was studied by immunohistochemistry, and the expression disparity in different clinic and pathologic staging of LHSCC was analyzed. The immunohistochemical staining of CD105 was done in the same microarray, the intratumor microvessel density (IMVD) was calculated by CD105 staining. The relationship between integrin alpha-v subunit expression and the IMVD was analyzed. RESULTS: In primary cancer tissue, the expression ratio of integrin alpha-v subunit was 68.0% (51/75), significantly higher than normal tissue beside cancer (10.3%, 3/29, chi2 = 28.68, P < 0.001); the expression ratio of integrin alpha-v subunit in lymph node metastatic carcinoma was 100.00% (20/20), significantly higher than normal tissue (chi2 = 38.77, P < 0.001) and primary cancer tissue (chi2 = 12.69, P < 0.05); in group with lymph node metastasis, the expression ratio of integrin alpha-v subunit was significantly higher than group without lymph node metastasis (chi2 = 10.87, P < 0.001); the IMVD of the group with integrin alpha-v subunit positive expression was significantly higher than the group with integrin alpha-v subunit negative expression (P < 0.001). CONCLUSIONS: There was significant relationship between integrin alpha-v subunit expression and lymphatic metastasis of LHSCC. Overexpression of the integrin alpha-v subunit may have contributed to the tumor angiogenesis and lead to lymphatic metastasis. Integrin alpha-v subunit may become a novel lymphatic metastasis marker of LHSCC.

[Experimental investigation of effects of adenovirus transduced tissue factor pathway inhibitor-2 gene on growth of laryngeal squamous carcinoma].

OBJECTIVE: To investigate the inhibitory effects of adenovirus transduced TFPI-2 gene on the growth of laryngeal squamous carcinoma. METHODS: Recombinant adenoviruses carrying human TFPI-2 gene were amplified and identified. The nude mouse model of laryngeal squamous carcinoma was established by intracutaneous injection of Hep-2 cells. Mice in the treated group were injected with recombinant adenoviruses with Ad-TFPI-2 (adenoviruses-TFPI-2) in peritumor tissue while mice in control group were injected with equivalent null plasmids. After treatment, the tumor weight and volume of tumor in each mouse were measured respectively. The morphological changes of tumor cells were observed using transmission electron microscope and proliferating cell nuclear antigen (PCNA) expression was examined using immunohistochemistry. RESULTS: The Ad-TFPI-2 virus titer was 2.8 x 10(12) PFU/ml after amplification. The average tumor weight and volume in Ad-TFPI-2 treated group were (1.20 +/- 0.34) cm3 and the volume (1.52 +/- 0.39) g, which were significantly lower than the tumor weight (2. 08 +/- 0.52) cm3 and (2.67 +/- 0.47) g in the control group (P < 0. 01). Apoptosis was observed in the tumors of Ad-TFPI-2 treated group. The PCNA index in Ad-TFPI-2 group was (54.9% +/- 12.4%), which was obviously lower than that (75.8% +/- 11.2%)in control group (P < 0.01). CONCLUSION: Peritumor injection of Ad-TFPI-2 can inhibit the growth of laryngeal squamous carcinoma in nude mouse model.

[Laryngeal function reconstruction with sternohyvoid muscle after partial laryngectomy].

OBJECTIVE: To study the laryngeal function reconstruction with sternohyvoid muscle after partial laryngectomy to treat T2 phase of glottis cancer. METHODS: 66 patients diagnosed to be at T2 phase of glottis cancer were treated by partial laryngectomy from 1992 to 1998. At the same time, they were rebuilt vocal cords with sternohyvoid muscle. RESULTS: All patients were decannulated from 2 to 3 weeks. They renewed own respiration, swallow and voice function. Overall three-year survival rate was 97.0% and five-year survival rate was 93.8%. CONCLUSION: New operation is superior than traditional one in improving survival rate and life quality.

[Relationship of matrilysin with invasion, metastasis, and prognosis in laryngeal cancer].

BACKGROUND & OBJECTIVE: Matrilysin (matrix metalloproteinases-7, MMP-7) might play an important role in invasion and metastasis of tumor and is thought to be related to prognosis of many kinds of carcinomas. This study was designed to investigate the significance of MMP-7 expression in the invasion and metastasis of laryngeal cancer and the relationship of MMP-7 expression with the prognosis of laryngeal cancer. METHODS: The expression of MMP-7 in 70 samples with paraffin-embedded laryngeal cancer and corresponding para-tumor normal mucosas were using immunohistochemical technique. The mRNA expression of MMP-7 were examined by reverse transcription-polymerase chain reaction (RT-PCR) in 24 frozen specimens of laryngeal cancer and corresponding para-tumor normal mucosas. RESULTS: (1) The immunohistochemical staining of MMP-7 was observed in 77.1% (54/70) of cancer tissues, which was significantly higher than that (5.7%, 4/70) of para-tumor normal mucosas (P< 0.01). The mRNA expression of MMP-7 was detected in 75.0% (18/24) of cancer tissues, which was also significantly higher than that (4.2%, 1/24) of para-tumor normal mucosas (P< 0.01). (2) The expression of MMP-7 in protein level was hot only higher in T3-T4 group than that in T1-T2 group (P< 0.01) but also higher in nodal metastases group than that in group without nodal metastases (P< 0.01). (3) The mRNA expression of MMP-7 was significantly higher in T3-T4 group than that in T1-T2 group (2.908 +/- 0.891 vs.1.662 +/- 0.508, P< 0.01) and was significantly higher in nodal metastases group than that in group without nodal metastases (2.958 +/- 0.827 vs. 1.887 +/- 0.769,P< 0.01). (4) Kaplan-Meier survival curve showed that the prognosis of group with MMP-7 protein high expression was poorer than that in group of MMP-7 protein weak or negative expression (log-rank=4.7559, 8.9513; P=0.0292, 0.0028). No significant difference was found between group with MMP-7 protein weak expression and group with that negative expression (log-rank=0.0314, P= 0.8593). CONCLUSION: MMP-7 possesses close relationship with the invasion, metastasis, and prognosis of laryngeal cancer, and it may be served as a marker in estimating the invasive and metastatic potency and prognosis of laryngeal cancer.

[Endostatin in the treatment of the transplantable model of human laryngeal squamous carcinoma in nude mice].

OBJECTIVE: To evaluate the inhibitory effect of endostatin on tumor growth of human laryngeal squamous carcinoma in nude mice and to explore the possible mechanism of the inhibition and the possible way of biological therapy. METHODS: Nude mice model bearing laryngocarcinoma was established by using human laryngeal squamous carcinoma cell line ( Hep-II). The animals were given endostatin (20 mg x kg(-1) x d(-1)) or PBS, for 21 consecutive days. The volumes of the subcutaneous tumor were observed. The microstructure in which the general 2-step immuohistochemical examination was adopted and ultra-microstructural changes of carcinoma after administration of endostatin were observed under light and electron microscopes for pathology examination. RESULTS: The differences were statistically significant for the net mice weight, tumor weight, and tumor volume and weight/net mice weight between the treatment group and the control group. The restrained percentage of tumor was 45.9%. The necrosis and apoptosis of the tumor cell and the angiogenesis reduction were found under light and electron microscope in the treatment group. The expression of MVD, PCNA and VEGF of the treatment group is lower than that of the control group, and T test showed that P < 0.01, P < 0.05, P < 0.05 respectively, the differences were statistically significant. CONCLUSIONS: These studies showed that endostatin could significantly restrain the development of laryngocarcinoma. The mechanism may be due to the effect of antiangiogesis.