Sanguinarine-dependent induction of apoptosis in primary effusion lymphoma cells.
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Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. In efforts to identify novel approaches to block proliferation of PEL cells, we found that sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, inhibits cell proliferation and induces apoptosis in a dose-dependent manner in several PEL cell lines. Our data show that sanguinarine treatment of PEL cells results in up-regulation of death receptor 5 (DR5) expression via generation of reactive oxygen species (ROS) and causes activation of caspase-8 and truncation of Bid (tBid). Subsequently, tBid translocates to the mitochondria causing conformational changes in Bax, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. Sanguinarine-induced release of cytochrome c results in activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, leading to induction of caspase-dependent apoptosis. In addition, we show that pretreatment of PEL cells with carbobenzoxy-Val-Ala-Asp-fluoromethylketone, a universal inhibitor of caspases, abrogates caspase and PARP activation and prevents cell death induced by sanguinarine. Moreover, treatment of PEL cells with sanguinarine down-regulates expression of inhibitor of apoptosis proteins (IAP). Finally, N-acetylcysteine, an inhibitor of ROS, inhibits sanguinarine-induced generation of ROS, up-regulation of DR5, Bax conformational changes, activation of caspase-3, and down-regulation of IAPs. Taken together, our findings suggest that sanguinarine is a potent inducer of apoptosis of PEL cells via up-regulation of DR5 and raise the possibility that this agent may be of value in the development of novel therapeutic approaches for the treatment of PEL. (+info)
Identification of novel regulators of apoptosis using a high-throughput cell-based screen.
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High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity. (+info)
Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse.
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Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of alpha1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if, rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple-helical collagen from the rib cartilage of cho/cho mice identified alpha1(V) and alpha2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie Blue staining of SDS-PAGE gels, in addition to alpha1(II)/alpha3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of alpha1(V) and alpha1(XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an alpha1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage. (+info)
Ethyl 2- [N-m-chlorobenzyl- (2'-methyl)] anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01006) induces apoptosis in human cervical cancer HeLa cells.
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Human cervical cancer is potentially lethal, and therefore the development of effective and tolerable therapeutic options is vital. In the present study, the in vitro effect of the synthetized compound JOT01006 (C21H20C1NO4) on human cervical epithelioid carcinoma cell line (HeLa) was examined. The results demonstrated that JOT01006 induced morphological changes and cytotoxicity (decreased the percentage of viable cells) in a dose-dependent manner. JOT01006 induced apoptosis which was analyzed by flow cytometric methods and confirmed by DAPI staining and DNA fragmentation analyzed by DNA gel electrophoresis. JOT01006 also induced reactive oxygen species (ROS) overproduction before causing endoplasmic reticulum (ER) stress which was also confirmed by the increased levels of Grp78 and Gadd153. Western blotting was selected to demonstrate that JOT010006 promoted p53, Bak, PARP, caspase-3 levels and decreased the levels of Bcl-2 and Bcl-xL. Our results also showed that JOT01006 also promoted caspase-12 production followed by apoptosis. The results also showed that JOT01006 inhibited the migration of HeLa cells potentially through inhibition of MMP-2 and -9. (+info)
Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation.
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BACKGROUND: De-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target beta-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that, in addition to the canonical WNT/beta-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation. METHODS: Using the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; beta-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors, or blocking antibodies. RESULTS: Phospho-DVL and stabilized beta-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active beta-catenin levels, lowers ERK1/2 activity, blocks proliferation, and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1, and MCF-7 cells. The effects of WNT signaling are mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor. CONCLUSION: Our data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments. (+info)
Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line.
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AIM: To investigate the apoptosis-inducing effect of oridonin, a diterpenoid isolated from Rabdosia rubescens, in the human cervical carcinoma HeLa cell line. METHODS: A morphological analysis, nuclear condensation, and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting. RESULTS: Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt), the expression of forkhead box class O (FOXO) transcription factor, and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate- ribose polymerase cleavage. In addition, Z-D(OMe)-E(OMe)-V-D(OMe)- FMK (z-DEVD-fmk), an inhibitor of caspases, prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally, oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein. CONCLUSION: Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt, FOXO, and GSK3) in the HeLa cell line, inhibiting the proliferation and induction of caspase-dependent apoptosis. (+info)
A functional polymorphism in COL11A1, which encodes the alpha 1 chain of type XI collagen, is associated with susceptibility to lumbar disc herniation.
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Lumbar disc herniation (LDH), degeneration and herniation of the nucleus pulposus of the intervertebral disc (IVD) of the lumbar spine, is one of the most common musculoskeletal diseases. Its etiology and pathogenesis, however, remain unclear. Type XI collagen is important for cartilage collagen formation and for organization of the extracellular matrix. We identified an association between one of the type XI collagen genes, COL11A1, and LDH in Japanese populations. COL11A1, which encodes the alpha 1 chain of type XI collagen, was highly expressed in IVD, but its expression was decreased in the IVD of patients with LDH. The expression level was inversely correlated with the severity of disc degeneration. A single-nucleotide polymorphism (c.4603C-->T [rs1676486]) had the most significant association with LDH (P=3.3 x 10(-6)), and the transcript containing the disease-associated allele was decreased because of its decreased stability. These observations indicate that type XI collagen is critical for IVD metabolism and that its decrease is related to LDH. (+info)
Immunohistochemical localization of collagen type XI alpha1 and alpha2 chains in human colon tissue.
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In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three alpha chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and alpha1(XI) and alpha2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon. (+info)