Effects of forkhead box C2 on carcinogenesis and lymphatic metastasis in endometrial carcinoma.

We investigated the distribution of endometrial lymphatic vessels and expression of forkhead box C2 (FOXC2) in normal endometrium during menstrual cycle and in endometrial adenocarcinoma. Full-thickness uterine samples and endometrial adenocarcinoma samples were collected for immunohistochemical analysis using D2-40 and FOXC2 mouse monoclonal antibodies. The lymphatic vessel density (LVD) of the endometrium was significantly reduced compared with the myometrium during the cycle. Intra-tumoral LVD was significantly decreased in both stages of endometrioid adenocarcinoma compared with normal endometrium and myometrium. Intra-tumoral LVD significantly decreased from stage IA to stage IIIC. Peri-tumoral LVD for stage IA and stage IIIC tumors was significantly increased compared with normal endometrial LVD, but decreased compared with normal myometrial LVD. Stage IIIC showed increased peri-tumoral LVD when compared with stage IA. The positive rate of FOXC2 was 73.3% in proliferative endometrium and 80% in secretory endometrium. Secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium. Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrium, both in the epithelium and stroma. FOXC2 expression in the stroma significantly increased when pelvic and/or para-aotic lymph nodes were involved. FOXC2 was immunolocalized in low-risk endometrial carcinoma in endometrioid adenocarcinoma, but not in normal endometrium. Endometrial lymphatic vessels were located in normal endometrium and myometrium across the menstrual cycle and in intra-and peri-endometrioid adenocarcinoma, and increased in endometrial adenocarcinoma. Peri-tumoral lymphatics were associated with increased lymphatic metastasis. FOXC2 may be associated with the genesis of endometrial carcinoma and lymphangiogensis in endometrial adenocarcinoma in intra- and peri-tumoral lymphatics.

Enhancement of antitumor activity of cisplatin in human lung cancer cells by tumor suppressor FUS1.

FUS1 is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. We previously showed that restoration of FUS1 function in 3p21.3-deficient human non-small-cell lung cancer (NSCLC) cells significantly inhibited tumor cell growth in vitro and in vivo. In this study, we evaluated the combined effects of the tumor suppressor FUS1 and the chemotherapeutic drug cisplatin on tumor cell growth and apoptosis induction in NSCLC cells, and explored the molecular mechanism of their mutual action. Exogenous expression of FUS1 by nanoparticle-mediated gene transfer sensitized the response of NSCLC cells to cisplatin, resulting in a 4- to 6-fold increase in tumor-suppressing activity. A systemic treatment with a combination of FUS1-nanoparticles and cisplatin in a human H322 lung cancer orthotopic xenograft mouse model dramatically enhanced the therapeutic efficacy of cisplatin. We also found that the FUS1-enhanced chemosensitivity is associated with the downregulation of MDM2, accumulation of p53 and activation of the Apaf-1-dependent apoptosis pathway. Our results demonstrated an important role of FUS1 in modulating chemosensitivity of lung cancer cells, and suggested that a proper combination of molecular therapeutics such as the proapoptotic tumor suppressor FUS1 and the conventional chemotherapeutic drugs such as cisplatin may be an efficient treatment strategy for human lung cancer.

Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K.-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized to plasma membrane, and image overlay showed colocalization of COX-2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin-1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.

Polymorphism and genetic relatedness among wild and cultivated rice species determined by AP-PCR analysis.

We have applied the arbitrarily primed polymerase chain reaction (AP-PCR) technique to the analysis of the relationships among six japonica and indica cultivars, and four wild species of rice. Chosen were four primers of arbitrary sequence that gave multiple amplification products when rice DNA was used as template. Among a total of 50 bands scored, 44 were polymorphic, which was sufficient to distinguish the species used in this study. It is apparent from the comparisons of genetic distances that cultivated rice (Oryza sativa) exhibits closest molecular affinity to wild O. rufipogon, suggesting that the origin of cultivated rice is from O. rufipogon. In a dendrogram of cultivated rice and O. rufipogon from different regions, japonica and indica rice were most closely clustered with O. rufipogon from China and India, respectively. Japonica and indica subspecies showed closer affinity with O. rufipogon from different origins than with each other, supporting a hypothesis of multi-centers of the origin of rice cultivation.