Yinlin Ge

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Anticancer Properties and Mechanisms of Fucoidan on Mouse Breast Cancer In Vitro and In Vivo

Background Fucoidan is a sulfated polysaccharide derived from brown algae that has been reported to perform multiple biological activities, including antitumor activity. In this study, we examined the influence of crude fucoidan on mouse breast cancer in vitro and in vivo. Materials and Methods In vitro, fluorescent staining, flow cytometry and Western blot were performed to analyze apoptosis and vascular endothelial growth factor (VEGF) expression of mouse breast cancer 4T1 cells. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. The tumor volume and weight were measured. The number of apoptotic cells and microvascular density (MVD) in tumor tissues were assessed by TUNEL and CD34 immunostaining. Immunohistochemical assays and ELISA assay were used to detect the expression of VEGF in tissues. Results In vitro studies showed that crude fucoidan significantly decreased the viable number of 4T1 cells, induced apoptosis and down-regulated the expression of VEGF. The expression of Bcl-2 was decreased, and the ratio of Bcl-2 to Bax was significantly decreased. The expression of Survivin and phosphorylated extracellular signal regulated protein kinases (ERKs) was decreased. Cytochrome C was released from mitochondria into cytosol, and the cleaved Caspase-3 protein rose after fucoidan treatment. Intraperitoneal injection of fucoidan in breast cancer models reduced the tumor volume and weight. The enhanced antitumor efficacy was associated with decreased angiogenesis and increased induction of apoptosis. Conclusion These findings indicated that crude fucoidan inhibited mouse breast cancer growth in vitro and in vivo. These data suggest that fucoidan may serve as a potential therapeutic agent for breast cancer.

Decreased expression of the mannose 6- phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells

BackgroundLoss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies.MethodsIn this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells.ResultsOur results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with the ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis.ConclusionsThese results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth.

Effects of ghrelin on hypothalamic glucose responding neurons in rats

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) with potent stimulatory effects on food intake. The aim of the present study was to investigate the effects of ghrelin on neuronal activity of hypothalamic glucose responding neurons. Single unit discharges in the lateral hypothalamic area (LHA), the ventromedial hypothalamic nucleus (VMH), and the parvocellular part of the paraventricular nucleus(pPVN) were recorded extracellularly by means of four-barrel glass micropipettes in anesthetized rats. The activity of glucose-sensitive neurons (GSNs) in the LHA, pPVN, and of glucoreceptor neurons (GRNs) in the VMH modulated by administration of ghrelin was analyzed. In the LHA, the majority of GSNs (17/25) increased in frequency due to ghrelin. Whereas the majority of VMH-GRNs (27/33) and pPVN-GSNs (9/13) was inhibited. The responses to ghrelin were abolished by pretreatment of [D-Lys-3]-GHRP-6, ghrelin receptor antagonist. These data indicate that the glucose responding neurons in the LHA, VMH, and pPVN are also involved in the orexigenic actions of ghrelin in the hypothalamic circuits, although AgRP/NPY neurons in the arcuate nucleus (ARC) are the primary targets of ghrelin.

Gene transfer of the Caenorhabditis elegans n-3 fatty acid desaturase inhibits neuronal apoptosis.

Previous studies have shown that n‐3 polyunsaturated fatty acids (PUFAs) can exert an antiapoptotic effect on neurons. The present study was designed to investigate whether the Caenorhabditis elegans fat‐1 gene encoding an n‐3 fatty acid desaturase (an enzyme that converts n‐6 PUFAs to corresponding n‐3 PUFAs) can be expressed functionally in rat cortical neurons and whether its expression can change the ratio of n‐6 : n‐3 fatty acids in the cell membrane and exert an effect on neuronal apoptosis. Infection of primary rat cortical cultures with Ad‐fat‐1 resulted in high expression of the fat‐1 gene. Lipid analysis indicated a decrease in the ratio of n‐6 : n‐3 PUFAs from 5.9 : 1 in control cells, to 1.45 : 1 in cells expressing the n‐3 fatty acid desaturase. Accordingly, the levels of prostaglandin E2, an eicosanoid derived from n‐6 PUFA, were significantly lower in cells infected with Ad‐fat‐1 when compared with control cells. Finally, there was a significant inhibition of growth factor withdrawal‐induced apoptotic cell death in neurons expressing the fat‐1 gene. These results demonstrate that expression of the fat‐1 gene can inhibit apoptotic cell death in neurons and suggest that the change in the n‐6 : n‐3 fatty acid ratio may play a key role in this protective effect.

Docosahexaenoic acid inhibited the Wnt/β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo ☆

N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/β-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced down-regulation of β-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/β-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer.

Fucoidan inhibited 4T1 mouse breast cancer cell growth in vivo and in vitro via downregulation of Wnt/β-catenin signaling.

Fucoidan is a sulfated polysaccharide derived from brown algae and is known to possess anticancer properties. However, the relationship between fucoidan and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, mouse breast cancer cells (4T1) were exposed to fucoidan to investigate the relationship between fucoidan and the Wnt/β-catenin signaling pathway in vivo and in vitro. We found that fucoidan significantly inhibited cell growth, increased cell death, and induced G1 cell cycle arrest in 4T1 cells. Fucoidan also reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity. Furthermore, fucoidan downregulated the expression of downstream target genes such as c-myc, cyclin D1, and survivin. Intraperitoneal injection of fucoidan in tumor-bearing mice reduced the tumor volume and weight. Fucoidan induced aberrant downregulation of β-catenin in tumor tissues with a significant increase in apoptosis. Thus, our data suggested that fucoidan exerts its anticancer activity through downregulation of Wnt/β-catenin signaling. Fucoidan may be an effective therapy for the chemoprevention and treatment of mouse breast cancer.

Down-regulation of the M6P/IGF-II receptor increases cell proliferation and reduces apoptosis in neonatal rat cardiac myocytes

BackgroundThe mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) is a multi-functional protein that has been implicated in regulation of cell growth and apoptosis. Cardiac myocytes express relatively high levels of M6P/IGF2R, and cardiomyocyte apoptosis has been identified in a variety of cardiovascular disorders, such as myocardial infarction and heart failure. However, involvement of M6P/IGF2R in the pathogenesis of these conditions has not been determined. Thus, the objective of this study was to determine the role of M6P/IGF2R in regulation of cardiac myocyte growth and apoptosis.ResultsWe down-regulated the expression of M6P/IGF2R in neonatal rat cardiac myocytes and examined the effect on cell proliferation and apoptosis. Infection of neonatal cardiomyocytes with an adenovirus expressing a ribozyme targeted against the M6P/IGF2R significantly reduced the level of M6P/IGF2R mRNA, as determined by RT-PCR and Ribonuclease Protection Assay (RPA). M6P-containing protein binding and endocytosis as well as the M6P/IGF2R-mediated internalization of 125I-IGF-II were lower in the ribozyme-treated cells than the control myocytes, indicating that the number of functional M6P/IGF2R in the ribozyme treated cells was reduced. Accordingly, a marked increase in cell proliferation and a reduced cell susceptibility to hypoxia- and TNF-induced apoptosis were observed in the ribozyme-treated cells.ConclusionsThese findings suggest that M6P/IGF2R may play a role in regulation of cardiac myocyte growth and apoptosis. Down regulation of this gene in cardiac tissues might be a new approach to prevention of cell death or promotion of mitogenesis for certain heart diseases.

The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo.

We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000™, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.

Antitumor effects of Isatin on human neuroblastoma cell line (SH-SY5Y) and the related mechanism

The purpose of the study was to investigate the antitumor effects of Isatin and the related mechanism. Human neuroblastoma cells (SH-SY5Y) were exposed to Isatin at different concentrations for 48 h. Apoptotic features were demonstrated by means of nuclei staining with Hoechst 33258 and flow cytometry with propidium iodide (PI). Expressions of Bcl-2, Bax and vascular endothelial growth factor (VEGF) mRNA were analyzed via RT-PCR. Expressions of Bcl-2, Bax proteins and phosphorylated extracellular signal regulated protein kinases (ERKs, p42/p44) were analyzed via Western blot. Activation of caspase-3 was assayed by flow cytometry with anti-active caspase-3-McAb-PE. VEGF protein was determined by ELISA kits. And the results showed that apoptosis of SH-SY5Y cells were induced by Isatin in a dose-dependent manner. Expressions of Bcl-2, VEGF mRNA and Bcl-2, VEGF proteins were down-regulated, while expressions of Bax mRNA and Bax protein were not changed obviously. Expression of phosphorylated ERKs decreased, but the level of activated caspase-3 increased after treatment of Isatin. These results suggest that Isatin promotes the apoptosis of neuroblastoma cells, therefore, it might be a potential candidate for the treatment of neuroblastoma.