Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from <Emphasis Type="Italic">Loranthus yadoriki</Emphasis>

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.     

Identifying differentially expressed genes in meta-analysis via Bayesian model-based clustering

A Bayesian model-based clustering approach is proposed for identifying differentially expressed genes in meta-analysis. A Bayesian hierarchical model is used as a scientific tool for combining information from different studies, and a mixture prior is used to separate differentially expressed genes from non-differentially expressed genes. Posterior estimation of the parameters and missing observations are done by using a simple Markov chain Monte Carlo method. From the estimated mixture model, useful measure of significance of a test such as the Bayesian false discovery rate (FDR), the local FDR (Efron et al., 2001), and the integration-driven discovery rate (IDR; Choi et al., 2003) can be easily computed. The model-based approach is also compared with commonly used permutation methods, and it is shown that the model-based approach is superior to the permutation methods when there are excessive under-expressed genes compared to over-expressed genes or vice versa. The proposed method is applied to four publicly available prostate cancer gene expression data sets and simulated data sets.     

Mycoparasitism of Rhizoctonia solani by Endophytic Chaetomium spirale ND35: Ultrastructure and Cytochemistry of the Interaction

The interaction between endophytic biocontrol agent Chaetomium spirale ND35 and the soil‐borne plant pathogen Rhizoctonia solani was studied by light microscopy and transmission electron microscopy (TEM), as well as further investigated by gold cytochemistry to assess the potential role of cell wall degrading enzymes (CWDEs) during the mycoparasitic process. Macroscopic observations of fungal growth in dual cultures revealed that pathogen growth inhibition occurred soon after contact with the antagonist, followed by the overgrowth of C. spirale on the colony of R. solani. The coiling of C. spirale around R. solani and intracellular growth of the antagonist in its host occurred frequently. Moreover, in advanced stage of interaction between the antagonist and the pathogen, The growth and development of C. spirale were associated with highly morphological changes of the host fungal cell, characterized by retraction of plasma membrane and cytoplasm disorganization. Further, TEM investigations through localization by gold immunocytochemistry showed that contact between the two fungi was mediated by an amorphous β‐1,3‐glucan‐enriched matrix originating from cell wall of the antagonist C. spirale and sticking to its host surface. At the same time, the hemispherical wall appositions which were intensely labeled by the antibodies of β‐1, 3‐glucan in cell wall of R. solani were induced to form at sites of potential antagonist entry. However, the antagonist was capable of penetrating this barrier, indicating that β‐1,3‐glucanases were produced during the mycoparasitic process. Localization of N‐acetylglucosamine residues (chitin) with the gold‐labelled wheat germ agglutinin (WGA) implicated that chitinases might be involved in the CWD of R. solani in this antagonistic process as well. This report is the first evidence about mechanisms of the interactions between C. spirale and R. solani in ultrastructural and cytochemical aspects.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.     

Candidate Markers That Associate with Chemotherapy Resistance in Breast Cancer through the Study on Taxotere-Induced Damage to Tumor Microenvironment and Gene Expression Profiling of Carcinoma-Associated Fibroblasts (CAFs)

Recently, emerging evidence has suggested that carcinoma-associated fibroblasts (CAFs) could contribute to chemotherapy resistances in breast cancer treatment. The aim of this study is to compare the gene expression profiling of CAFs before and after chemotherapy and pick up candidate genes that might associate with chemotherapy resistance and could be used as predictors of treatment response. CAFs were cultured from surgically resected primary breast cancers and identified with immunohistochemistry (IHC) and Flow cytometry (FCM). MDA-MB-231 cells were cultured as the breast cancer cell line. Cell adhesion assay, invasion assay, and proliferation assay (MTT) were performed to compare the function of MDA-MB-231 cells co-cultured with CAFs and MDA-MB-231 cells without co-culture, after chemotherapy. Totally 6 pairs of CAFs were prepared for microarray analysis. Each pair of CAFs were obtained from the same patient and classified into two groups. One group was treated with Taxotere (regarded as after chemotherapy) while the other group was not processed with Taxotere (regarded as before chemotherapy). According to our study, the primary-cultured CAFs exhibited characteristic phenotype. After chemotherapy, MDA-MB-231 cells co-cultured with CAFs displayed increasing adhesion, invasiveness and proliferation abilities, compared with MDA-MB-231 cells without CAFs. Moreover, 35 differentially expressed genes (absolute fold change >2) were identified between CAFs after chemotherapy and before chemotherapy, including 17 up-regulated genes and 18 down-regulated genes. CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were picked up as the candidate markers, of which the differential expression in CAFs before and after chemotherapy was confirmed. The results indicate the changes of gene expression in CAFs induced by Taxotere treatment and propose the candidate markers that possibly associate with chemotherapy resistance in breast cancer.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.