Wood Yee Chan

SPARC (Secreted Protein Acidic and Rich in Cysteine) Induces Apoptosis in Ovarian Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular Ca(2+)-binding matricellular glycoprotein that associates with cell populations undergoing migration, morphogenesis, and differentiation. Studies on endothelial cells have established that its principal functions in vitro are counteradhesion and antiproliferation. The mechanism(s) underlying these antitumor effects is unknown. In this study, we showed that SPARC expression in ovarian cancer cells is inversely correlated with the degree of malignancy. The immunohistochemical data presented here confirmed the importance of diminished SPARC expression in ovarian cancer development. Treating human ovarian surface epithelial cells and ovarian cancer cells with SPARC revealed that as SPARC inhibits the proliferation of both normal and cancer cells, it induces apoptosis only in cancer cells. This observation indicates that down-regulation of SPARC is essential for ovarian carcinogenesis as cancer cells become sensitized to the apoptotic activity of SPARC during malignant transformation. We also showed here the first direct evidence that putative SPARC receptors are present on ovarian epithelial cells. Their levels are higher in human ovarian surface epithelial cells than cancer cells. Binding of SPARC to its receptor is likely to trigger tissue-specific signaling pathways that mediate its tumor suppressing functions. Decrease in ligand-receptor interaction by the down-regulation of SPARC and/or its receptor is essential for ovarian carcinogenesis.

Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design

The threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virus and the host cell. It, therefore, plays an important structural and functional role and represents an attractive drug target. Here, we report the 3.3‐Å crystal structure of H5N1 NP, which is composed of a head domain, a body domain, and a tail loop. Our structure resolves the important linker segments (residues 397–401, 429–437) that connect the tail loop with the remainder of the molecule and a flexible, basic loop (residues 73–91) located in an arginine‐rich groove surrounding Arg150. Using surface plasmon resonance, we found the basic loop and arginine‐rich groove, but mostly a protruding element containing Arg174 and Arg175, to be important in RNA binding by NP. We also used our crystal structure to build a ring‐shaped assembly of nine NP subunits to model the miniribonucleo‐protein particle previously visualized by electron microscopy. Our study of H5N1 NP provides insight into the oligomerization interface and the RNA‐binding groove, which are attractive drug targets, and it identifies the epitopes that might be used for universal vaccine development.—Ng, A. K.‐L., Zhang, H., Tan, K., Li, Z., Liu, J.‐h., Chan, P. K.‐S., Li, S.‐M., Chan, W.‐Y., Au, S. W.‐N., Joachimiak, A., Walz, T., Wang, J.‐H., Shaw, P.‐C. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB J. 22, 3638–3647 (2008)

DOC-2, a candidate tumor suppressor gene in human epithelial ovarian cancer

Using RNA fingerprinting (RAP) strategy and Northern blot analysis, we identified a differentially expressed sequence DOC-2 which is detectable in all normal human ovarian surface epithelial (HOSE) cell cultures but not in ovarian cancer cell lines and tissues. Subsequent cloning of DOC-2 from a cDNA library generated from the HOSE cells was carried out using the 3′ and 5′ RACE approach. A 3268 base pair full length cDNA of DOC-2 was isolated and sequenced. The predicted protein has a length of 770 amino acids. Homology search of all NCBI sequences indicated that the amino acid sequence of DOC-2 shares 93% homology with the mouse p96/mDab2 phosphoprotein and has a phosphotyrosine interacting domain (PID) and multiple SH3 binding motifs. Chromosomal localization by FISH showed that the DOC-2 gene is located on 5p13. Western blot analysis showed that the 105 kDa DOC-2 protein was down-regulated in all the carcinoma cell lines. In-situ immunohistochemistry performed on normal ovaries, and benign, borderline and invasive ovarian tumor tissues showed down regulation of DOC-2 protein particularly in serous ovarian tumor tissues. When DOC-2 was transfected into the ovarian carcinoma cell line SKOV3, the stable transfectants showed significantly reduced growth rate and ability to form tumors in nude mice. These data suggest that down-regulation of DOC-2 may play an important role in ovarian carcinogenesis.

Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants.

1. The biochemical characteristics and biological activities of eight Cucurbitaceae plant proteins designated trichosanthin (isolated from tubers of Trichosanthes kirilowii), beta-trichosanthin (isolated from tubers of Trichosanthes cucumeroides), alpha- and beta-momorcharins (isolated from seeds of Momordica charantia), momorchochin (isolated from tubers of Momordica cochinchinensis), luffaculin (isolated from seeds of Luffa acutangula) and luffin-a and luffin-b (isolated from seeds of Luffa cylindrica), were reviewed. 2. The isolation procedures for all eight proteins are based on aqueous extraction, acetone fractionation and ion exchange chromatography. Ammonium sulfate precipitation and gel filtration are steps which may be included to improve purification. 3. The proteins are basic in nature and possess a molecular weight of approx. 30,000. All except trichosanthin are glycoproteins. The content of Asx and Glx residues is high. The N-terminal amino acid residue is Asp. Their amino acid compositions and N-terminal amino acid sequences are similar. 4. Circular dichroism spectroscopic studies revealed that trichosanthin, alpha- and beta-momorcharins possess similar secondary but different tertiary structures. 5. Most of the proteins are immunologically distinct. 6. The proteins exhibit abortifacient, antitumor, ribosome inactivating and immunomodulatory activities. Trichosanthin manifests anti-human immunodeficiency virus activity.

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH(2)-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.

Bcl-2 and p53 Protein Expression, Apoptosis, and p53 Mutation in Human Epithelial Ovarian Cancers

Bcl-2 and p53 gene products have been both linked to cell death by apoptosis. In the present study, we examined the relationship of Bcl-2 and p53 protein expression, p53 mutation and apoptosis in normal human ovaries and different types of human ovarian epithelial tumors by immunohistochemical localization, in situ terminal transferase-mediated dUTP nick end labeling and polymerase chain reaction-single strand conformation polymorphism. It was found that Bcl-2 expressed strongly in the surface epithelium of normal ovaries and benign and borderline ovarian tumors but weakly in the malignant tumors. On the contrary, strong protein expression of p53 was found in 54% (25/46) of the malignant epithelial tumors examined but similar expression of p53 was not observed in borderline and benign tumors and normal ovarian surface epithelium. A significant inverse correlation between Bcl-2 and p53 expression was found in the malignant ovarian tumors examined. p53 gene mutation at exons 5-11 was however not a pre-requisite for p53 expression in both borderline and malignant tumors. Apoptotic activities, as reflected by apoptotic indices, were low in normal ovarian surface epithelium and benign tumors but were increased in borderline and malignant tumors, with the highest average apoptotic index found in grade III malignant tumors. Statistical analyses showed a positive correlation between apoptosis and p53 expression, but similar correlation was not found between apoptosis and Bcl-2 expression. Our results also indicate that although expression of Bcl-2 is important during ovarian carcinogenesis, the Bcl-2 protein may have other roles to play apart from being a modulator of apoptosis in human ovarian epithelial cancers.

Thrombopoietin Protects Against In Vitro and In Vivo Cardiotoxicity Induced by Doxorubicin

Background— Doxorubicin (DOX) is an important antineoplastic agent. However, the associated cardiotoxicity, possibly mediated by the production of reactive oxygen species, has remained a significant and dose-limiting clinical problem. Our hypothesis is that the hematopoietic/megakaryocytopoietic growth factor thrombopoietin (TPO) protects against DOX-induced cardiotoxicity and might involve antiapoptotic mechanism exerted on cardiomyocytes. Methods and Results— In vitro investigations on H9C2 cell line and spontaneously beating cells of primary, neonatal rat ventricle, as well as an in vivo study in a mouse model of DOX-induced acute cardiomyopathy, were performed. Our results showed that pretreatment with TPO significantly increased viability of DOX-injured H9C2 cells and beating rates of neonatal myocytes, with effects similar to those of dexrazoxane, a clinically approved cardiac protective agent. TPO ameliorated DOX-induced apoptosis of H9C2 cells as demonstrated by assays of annexin V, active caspase-3, and mitochondrial membrane potential. In the mouse model, administration of TPO (12.5 &mgr;g/kg IP for 3 alternate days) significantly reduced DOX-induced (20 mg/kg) cardiotoxicity, including low blood cell count, cardiomyocyte lesions (apoptosis, vacuolization, and myofibrillar loss), and animal mortality. Using Doppler echocardiography, we observed increased heart rate, fractional shortening, and cardiac output in animals pretreated with TPO compared with those receiving DOX alone. Conclusions— These data have provided the first evidence that TPO is a protective agent against DOX-induced cardiac injury. We propose to further explore an integrated program, incorporating TPO with other protocols, for treatment of DOX-induced cardiotoxicity and other forms of cardiomyopathy.

Proliferation and apoptosis in the developing human neocortex

The cell kinetics of the developing central nervous system (CNS) is determined by both proliferation and apoptosis. In the human neocortex at week 6 of gestation, proliferation is confined to the ventricular zone, where mitotic figures and nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA) are detectable. Cell division is symmetric, with both daughter cells reentering mitosis. At week 7, the subventricular zone, a secondary proliferative zone, appears. It mainly gives rise to local circuit neurons and glial cells. Around week 12, the ventricular and subventricular zones are thickest, and the nuclear PCNA label is strongest, indicating that proliferation peaks at this stage. Thereafter, asymmetric division becomes the predominant mode of proliferation, with one daughter cell reentering mitosis and the other one migrating out. Towards late gestation, the ventricular and subventricular zones almost completely disappear and proliferation shifts towards the intermediate and subplate zones, where mainly glial cells are generated. A remnant of the subventricular zone with proliferative activity persists into adulthood. In general, proliferation follows a latero‐medial gradient in the neocortex lasting longer in its lateral parts. Apoptotic nuclei have been detected around week 5, occurring in low numbers in the ventricular zone at this stage. Apoptotic cell death increases around midgestation and then spreads throughout all cortical layers, with most dying cells located in the ventricular and subventricular zones. This spatial distribution of apoptosis extends into late gestation. During the early postnatal period, most apoptotic cells are still located in the subcortical layers. During early embryonic development, proliferation and apoptosis are closely related, and are probably regulated by common regulators. In the late fetal and early postnatal periods, when proliferation has considerably declined in all cortical layers, apoptosis may occur in neurons whose sprouting axons do not find their targets. Anat Rec 267:261–276, 2002.

Bone marrow mesenchymal stem cells in a three-dimensional gelatin sponge scaffold attenuate inflammation, promote angiogenesis, and reduce cavity formation in experimental spinal cord injury.

Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a thin film of poly-(lactide-co-glycolide) (PLGA). Rat bone marrow-derived mesenchymal stem cells (MSCs) were isolated, cultured, and then seeded to the scaffolds. Distribution of cells and cell growth, survival, and proliferation within the scaffolds were then determined. Immunofluorescence and Western blot analysis were employed to detect the deposition of fibronectin to the scaffolds on day 3 and day 7 of culture. Scaffolds with or without MSCs were then transplanted into the transected rat spinal cord. One or 8 weeks following transplantation, cavity areas, activated macrophages/microglia, expression of TNF-α and IL-1β, and neovascularization within the grafts were examined and quantified. Deposition of fibronectin (FN) and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) as potential inducing factors for angiogenesis were also examined. Results showed that 3D GS scaffolds allowed MSCs to adhere, survive, and proliferate and also FN to deposit. In vivo transplantation experiments demonstrated that these scaffolds were biocompatible, and MSCs seeded to the scaffolds played an important role in attenuating inflammation, promoting angiogenesis, and reducing cavity formation. Therefore, the GS scaffolds with MSCs may serve as promising supporting transplants for repairing spinal cord injury.

Expression of P2X purinoceptors during rat brain development and their inhibitory role on motor axon outgrowth in neural tube explant cultures

Extracellular ATP is well known as a neurotransmitter and neuromodulator in the CNS of adults. However, little is known about the involvement of ATP during the development of mammalian brain. In the present study, we have examined the expression pattern of P2X receptor subtype mRNA and protein during perinatal rat brain development (from embryonic day (E) 10 to postnatal day (P) 16 brain). While P2X3 receptors appeared early at E11, they declined in the stages that follow. P2X2 and P2X7 receptors were expressed from E14 onwards, while P2X4, P2X5 and P2X6 receptors were expressed from P1 onwards. P2X1 receptor expression was not observed in any of the developmental ages examined. We investigated the effect of 100 microM ATP and alpha,beta-methylene ATP (alpha,beta-meATP; selective agonist for P2X1, P2X2/3 and P2X3 receptors) on motor axon outgrowth in collagen-embedded neural tube explant cultures. Both ATP- and alpha,beta-meATP-treated neural tubes showed a significant reduction in neurite outgrowth compared with the control explants. This inhibitory effect could not be reproduced by uridine triphosphate. In conclusion, all P2X receptor subtypes, except for P2X1, were strongly represented in the developing rat brain. ATP was shown to inhibit motor axon outgrowth during early embryonic neurogenesis, most likely via the P2X3 receptor. It is speculated that P2X7 receptors may be involved in programmed cell death during embryogenesis and that P2X4, P2X(5) and P2X6 receptors might be involved in postnatal neurogenesis.