Rong-Hong Hsieh

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60–95% reduction in Tfam gene expression and 50–90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA‐encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)‐encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus‐like to a spindle‐like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a 4,977‐bp deletion, and a 3,243‐point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. J. Cell. Biochem. 103: 347–357, 2008.

Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression

Gastric carcinoma is one of the most common malignancies and a lethal cancer in the world. Notch signaling and transcription factors STAT3 (signal transducer and activator of transcription 3) and Twist regulate tumor development and are critical regulators of gastric cancer progression. Herein, the relationship among Notch, STAT3 and Twist pathways in the control of gastric cancer progression was studied. We found that Twist and phosphorylated STAT3 levels were promoted by the activated Notch1 receptor in human stomach adenocarcinoma SC-M1, embryonic kidney HEK293 and erythroleukemia K562 cells. Notch1 signaling dramatically induced Twist promoter activity through a C promoter binding factor-1-independent manner and STAT3 phosphorylation. Overexpression of Notch1 receptor intracellular domain (N1IC) enhanced the interaction between nuclear STAT3 and Twist promoter in cells. Gastric cancer progression of SC-M1 cells was promoted by N1IC through STAT3 phosphorylation and Twist expression including colony formation, migration and invasion. STAT3 regulated gastric cancer progression of SC-M1 cells via Twist. N1IC also elevated the progression of other gastric cancer cells such as AGS and KATO III cells through STAT3 and Twist. The N1IC-promoted tumor growth and lung metastasis of SC-M1 cells in mice were suppressed by the STAT3 inhibitor JSI-124 and Twist knockdown. Furthermore, Notch1 and Notch ligand Jagged1 expressions were significantly associated with phosphorylated STAT3 and Twist levels in gastric cancer tissues of patients. Taken together, these results suggest that Notch1/STAT3/Twist signaling axis is involved in progression of human gastric cancer and modulation of this cascade has potential for the targeted combination therapy.

Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos

Abstract: To clarify the relationship between mitochondria and embryo development, we collected human unfertilized oocytes, early embryos, and arrested embryos. Unfertilized oocytes and poor‐quality embryos were collected, and the ultrastructure of mitochondria was determined by transmission electron micrography. Four criteria for determining the mitochondrial state were mitochondrial morphology, cristae shape, location, and number of mitochondria. In mature oocytes, mitochondria were rounded with arched cristae and a dense matrix and were distributed evenly in the ooplasm. In pronuclear zygotes, the size and shape of mitochondria were similar to those in mature oocytes; however, mitochondria appeared to migrate and concentrate around pronuclei. In this study, 67% of examined unfertilized oocytes had fewer mitochondria in the cytoplasm. A decreased number of mitochondria located near the nucleus was also demonstrated in 60% of arrested embryos. Fewer differentiated cristae were determined in all three arrested blastocyst stages of embryos. The relative expressions of oxidative phosphorylation genes in oocytes and embryos were also determined. These data imply that inadequate redistribution of mitochondria, unsuccessful mitochondrial differentiation, or decreased mitochondrial transcription may result in poor oocyte fertilization and compromised embryo development.

The Activated Notch1 Receptor Cooperates with α-Enolase and MBP-1 in Modulating c-myc Activity

ABSTRACT The Notch signal pathway plays multifaceted roles to promote or suppress tumorigenesis. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, activates the c-myc proto-oncogene. The complex of N1IC and transcription factor YY1 binds to the human c-myc promoter to enhance c-myc expression in a CBF1-independent manner. Here we demonstrated that N1IC interacted with the c-Myc-regulating proteins α-enolase and c-myc promoter binding protein 1 (MBP-1). Both α-enolase and MBP-1 suppressed the N1IC-enhanced activity of the c-myc promoter in a CBF1-independent manner. The YY1 response element in front of the P2 c-myc promoter was essential and sufficient for the modulation of c-myc by N1IC and α-enolase or MBP-1. Furthermore, N1IC, YY1, and α-enolase or MBP-1 but not CBF1 bound to the c-myc promoter through associating with the YY1 response element. Hemin-induced erythroid differentiation was suppressed by N1IC in K562 cells. This suppression was relieved by the expression of α-enolase and MBP-1. In addition, both α-enolase and MBP-1 suppressed the N1IC-enhanced colony-forming ability through c-myc. These results indicate that the activated Notch1 receptor and α-enolase or MBP-1 cooperate in controlling c-myc expression through binding the YY1 response element of the c-myc promoter to regulate tumorigenesis.

Notch2‐induced COX‐2 expression enhancing gastric cancer progression

Gastric carcinoma is one of the most common and mortal types of malignancy worldwide. To date, the mechanisms controlling its aggressiveness are not yet fully understood. Notch signal pathway can function as either an oncogene or a tumor suppressor in tumorigenesis. Four members (Notch1–4) of Notch receptors were found in mammals and each exhibits distinct roles in tumor progression. Previous study showed that the activated Notch1 receptor promoted gastric cancer progression through cyclooxygenase‐2 (COX‐2). This study addressed whether Notch2 signal pathway is also involved in gastric cancer progression. Constitutive expression of Notch2 intracellular domain (N2IC), the activated form of Notch2 receptor, promoted both cell proliferation and xenografted tumor growth of human stomach adenocarcinoma SC‐M1 cells. The colony formation, migration, invasion, and wound‐healing abilities of SC‐M1 cells were enhanced by N2IC expression, whereas these abilities were suppressed by Notch2 knockdown. Similarly, Notch2 knockdown inhibited cancer progressions of AGS and AZ521 gastric cancer cells. Expression of N2IC also caused epithelial–mesenchymal transition in SC‐M1 cells. Furthermore, N2IC bound to COX‐2 promoter and induced COX‐2 expression through a CBF1‐dependent manner in SC‐M1 cells. The ability of N2IC to enhance tumor progression in SC‐M1 cells was suppressed by knockdown of COX‐2 or treatment with NS‐398, a COX‐2 inhibitor. Moreover, the suppression of tumor progression by Notch2 knockdown in SC‐M1 cells was reversed by exogenous COX‐2 or its major enzymatic product PGE2. Taken together, this study is the first to demonstrate that the Notch2‐COX‐2 signaling axis plays an important role in controlling gastric cancer progression.

MBP-1 suppresses growth and metastasis of gastric cancer cells through COX-2.

The c-Myc promoter binding protein 1 (MBP-1) is a transcriptional suppressor of c-myc expression and involved in control of tumorigenesis. Gastric cancer is one of the most frequent neoplasms and lethal malignancies worldwide. So far, the regulatory mechanism of its aggressiveness has not been clearly characterized. Here we studied roles of MBP-1 in gastric cancer progression. We found that cell proliferation was inhibited by MBP-1 overexpression in human stomach adenocarcinoma SC-M1 cells. Colony formation, migration, and invasion abilities of SC-M1 cells were suppressed by MBP-1 overexpression but promoted by MBP-1 knockdown. Furthermore, the xenografted tumor growth of SC-M1 cells was suppressed by MBP-1 overexpression. Metastasis in lungs of mice was inhibited by MBP-1 after tail vein injection with SC-M1 cells. MBP-1 also suppressed epithelial-mesenchymal transition in SC-M1 cells. Additionally, MBP-1 bound on cyclooxygenase 2 (COX-2) promoter and downregulated COX-2 expression. The MBP-1-suppressed tumor progression in SC-M1 cells were through inhibition of COX-2 expression. MBP-1 also exerted a suppressive effect on tumor progression of other gastric cancer cells such as AGS and NUGC-3 cells. Taken together, these results suggest that MBP-1-suppressed COX-2 expression plays an important role in the inhibition of growth and progression of gastric cancer.

Th17 Cells as Potential Probiotic Therapeutic Targets in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are characterized by wasting and chronic intestinal inflammation triggered by various cytokine-mediated pathways. In recent years, it was shown that T helper 17 (Th17) cells are involved in the pathogenesis of IBD, which makes them an attractive therapeutic target. Th17 cells preferentially produce interleukin (IL)-17A–F as signature cytokines. The role of the interplay between host genetics and intestinal microbiota in the pathogenesis of IBD was demonstrated. Probiotics are live microorganisms that when orally ingested in adequate amounts, confer a health benefit to the host by modulating the enteric flora or by stimulating the local immune system. Several studies indicated the effectiveness of probiotics in preventing and treating IBD (ulcerative colitis, and Crohn’s disease). Furthermore, there is mounting evidence of probiotics selectively targeting the Th17 lineage in the prevention and management of inflammatory and autoimmune diseases such as IBD. This review highlights critical roles of Th17 cells in the pathogenesis of IBD and the rationale for using probiotics as a novel therapeutic approach for IBD through manipulation of Th17 cells. The potential molecular mechanisms by which probiotics modulate Th17 cells differentiation and production are also discussed.

Nuclear βII-Tubulin Associates with the Activated Notch Receptor to Modulate Notch Signaling

The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1–dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear βII-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of βII-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of βII-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of α- and βII-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with βII-tubulin. Furthermore, α- and βII-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear α- and βII-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1–dependent transactivation activity of N1IC. We also show that nuclear βII-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear βII-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.

Notch1 pathway-mediated microRNA-151-5p promotes gastric cancer progression

Gastric carcinoma is the third leading cause of lethal cancer worldwide. Previous studies showed that Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor, promotes gastric cancer progression. It has been demonstrated that a significant cross-talk interplays between Notch pathways and microRNAs (miRNAs) in controlling tumorigenesis. This study identified an intronic microRNA-151 (miR-151), which consists of two mature miRNAs, miR-151-3p and miR-151-5p, as a Notch1 receptor-induced miRNA in gastric cancer cells. Activation of Notch1 pathway enhanced expressions of miR-151 and its host gene, focal adhesion kinase (FAK), in gastric cancer cells. The levels of miR-151 in gastric cancer samples were higher than those of adjacent non-tumor samples. Activated Notch1 pathway induced CBF1-dependent FAK promoter activity. The ectopic expression of miR-151 promoted growth and progression of SC-M1 gastric cancer cells including cell viability and colony formation, migration, and invasion abilities. Activated Notch1 pathway could augment progression of gastric cancer cells through miR-151-5p and FAK. The mRNA levels of pluripotency genes, Nanog and SOX-2, tumorsphere formation ability, tumor growth, and lung metastasis of SC-M1 cells were elevated by activated Notch1 pathway through miR-151-5p. Furthermore, miR-151-5p could target 3′-untranslated region (3′-UTR) of p53 mRNA and down-regulate p53 level in SC-M1 cells. Mechanistically, Notch1/miR-151-5p axis contributed to progression of SC-M1 cells through down-regulation of p53 which in turn repressed FAK promoter activity. Taken together, these results suggest that Notch1 pathway and miR-151-5p interplay with p53 in a reciprocal regulation loop in controlling gastric carcinogenesis.

Linoleic acid promotes mitochondrial biogenesis and maintains mitochondrial structure for prevention of streptozotocin damage in RIN-m5F cells

Linoleic acid (LA) improves insulin resistance and prevents diabetes. To investigate whether linoleic acid could protect against streptozotocin (STZ)-induced cell death, rat RIN-m5F cells were exposed to STZ. SL and SO groups consisted of cells treated with STZ and then LA or oleic acid (OA) respectively. STZ treatment decreased the mitochondrial membrane potential in the STZ, SO, and SL groups. Cells of the SL group had more intact mitochondria. Increased mRNA expression of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), as well as of the mitochondrial biogenesis regulators peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), and mitochondrial transcription factor A (Tfam), were found in the LA group. The insulin content was significantly decreased in all three groups. These results suggest that the effects of LA on cell viability after STZ damage occur through maintenance of mitochondrial structure and increased mitochondrial biogenesis.