Fuxue Chen

Down-regulation of 67LR reduces the migratory activity of human glioma cells in vitro.

OBJECTIVES Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Experimental evidence indicates that the 67 kDa elastin-laminin receptor (67LR) subunit is a high-affinity non-integrin laminin-binding protein that is over-expressed on the tumor cell surface in a variety of human carcinomas, and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize. However, little is known of the expression and function of 67LR in glioma cells. METHODS In this study, we estimated whether 67LR was constitutively over-expressed in high-grade astrocytomas by immunohistochemical staining and Western blotting, and investigated the role of a low level of 67LR expression in glioma cell line-U251 by constructing an interfering RNA expression plasmid. RESULTS The results showed that the 67LR had an enhanced over-expression in high-grade astrocytomas against normal brain tissues samples, and that the migratory activity of glioma cells was reduced after the down-regulation of the 67LR gene by RNAi. DISCUSSION It was hypothesized that a low level of 67LR expression could reduce migratory activity of glioma cells, which further proved that 67LR played an important role in glioma invasion by mediating tumor cell functions leading to sarcomata. This study provided a new alternative to gene therapy for glioma treatment.

Epigallocatechin-3-gallate induces apoptosis, inhibits proliferation and decreases invasion of glioma cell.

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been considered a potential therapeutic and chemopreventive agent for cancer. Glioma is a malignant tumor with high mortality but effective therapy has not yet been developed. In this study, we found that EGCG induced apoptosis in U251 glioma cells via the laminin receptor (molecular weight 67kDa) in a time- and dose-dependent manner, decreased their invasiveness and inhibited their proliferation. The mitogen-activated protein kinase pathway was shown to be involved in glioma cell apoptosis and proliferation. Furthermore, the mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9 were reduced after EGCG treatment. These results suggest that EGCG has important therapeutic effects with low toxicity and side-effects, and could be used in cancer chemoprevention.

Down-regulation of Stat3 induces apoptosis of human glioma cell: a potential method to treat brain cancer

Abstract Objectives: Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Stat3 is a member of the signal transducer and activator of transcription family, and it has the potential to mediate cell survival, growth and differentiation. Methods: In this study, we testified that Stat3 was constitutively expressed in glioma cell line SHG44 and then investigated the role of a low level of Stat3 expression in glioma cells by constructing an interfering RNA expression plasmid. Results: The results showed that glioma cells underwent morphologic and biochemical changes after the RNAi treatment. Discussion: We hypothesized that a low level of Stat3 expression could induce apoptosis of glioma cell, which further proved that Stat3 played an important role in growth, survival and proliferation of glioma cells. This study provides a new alternative to gene therapy for glioma treatment.

Expression of bitter taste receptor Tas2r105 in mouse kidney.

The kidney is the most important excretory organ in the body and plays an essential role in maintaining homeostasis in vivo by conserving body fluid and electrolytes and removing metabolic waste. In this study, three types of transgenic system were used to investigate the expression of the bitter taste receptor Tas2r105 in mouse renal tissue (Tas2r105-GFP/Cre, Tas2r105-GFP/Cre-DTA and Tas2r105-GFP/Cre-LacZ). The results suggest that bitter taste receptors Tas2r105 and Tas2r106 are expressed in the renal corpuscle and the renal tubule, including the proximal tubule and distal tubule. Expression of α-gustducin, an important component of taste signal transduction, was also detected in mouse kidney. Meanwhile, conditional diphtheria toxin (DTA) expression in Tas2r105+ cells caused an increase in size of the glomerulus and renal tubule, accompanied by a decrease in cell density in the glomerulus. This indicates that Tas2r105+ cells play an important role in maintaining the structure of the glomerulus and renal tubules. Overall, the current study collectively demonstrates that cells labeled by bitter taste receptor expression may play a critical role in controlling human health, and have properties far beyond the original concept of taste perception.

Targeting Stat3 suppresses growth of U251 cell-derived tumours in nude mice

Malignant gliomas are highly invasive tumours associated with high levels of mortality, and the treatment of gliomas remains a major neurosurgical challenge. Stat3, a member of the signal transducer and activator of transcription family, has a critical role in a variety of cancer cells. We have previously shown that downregulation of Stat3 decreases invasiveness and induces apoptosis in U251 human glioma cells in vitro, but to date it has been unclear whether this treatment would be beneficial in vivo. In the present study, we found that downregulation of Stat3 via RNAi suppressed tumour growth in a xenograft mouse model by inducing apoptosis of U251 tumour cells and inhibiting tumour neo-angiogenesis. We also found that Stat3 RNAi suppresses the expression of Bcl-2 in vivo to induce apoptosis. These results indicate that Stat3 is a critical factor in the survival of patients with glioma, and that targeting Stat3 may offer a potential therapeutic approach.

Inhibition of Stat3 expression induces apoptosis and suppresses proliferation in human leukemia HL-60 cells

Abstract Acute myeloid leukemia (AML) is the most common myeloid leukemia. It is highly malignant, thus, most patients with AML will relapse and die after traditional treatment. Stat3, a member of the signal transducer and activator of transcription (Stat) family, is involved in the development and progression of many tumors. The purpose of the study was to investigate whether the down-regulation of Stat3 expression by RNA interference is effective against human leukemia HL-60 cells. The results indicated that constitutively expressed Stat3 is present in human leukemia HL-60 cells, and the down-regulation of Stat3 expression caused significant induction of apoptosis as well as inhibition of proliferation in HL-60 cells. These data further demonstrated that Stat3 plays a critical role in human leukemia HL-60 cell apoptosis and proliferation. Thus, targeting Stat3 may be a useful adjunctive treatment strategy in AML.

An identification of stem cell-resembling gene expression profiles in high-grade astrocytomas†

High‐grade astrocytomas are among the most intractable types of cancers and are often fatal. Previous studies have suggested that high‐grade astrocytomas may adopt the self‐renewal and migration properties of neural stem cells (NSCs) to proliferate and spread by expressing the stem cell‐specific genes. However, despite a few common molecules being documented, the molecular basis underlying these similarities remains largely unknown. To have a better understanding of the stem cell characteristics of high‐grade astrocytomas, we performed the study to identify the stem cell‐resembling gene expression profile in high‐grade astrocytomas. cDNA microarray analysis was used to detect the differentially expressed genes of isolated human high‐grade astrocytomas versus their peritumoral tissue counterparts, and the identification of stem cell‐resembling genes was approached by comparing the high‐grade astrocytomas‐specific gene expression profile with that of NSCs identified by our previous study and other groups. We identified more than 200 high‐grade astrocytomas‐specific genes in this study, and near 10% genes or gene families of them exhibited similar up or down expression patterns as in NSCs. Further analysis indicated that these genes were actively involved in cell proliferation, adhesion, migration, and metastasis. This study revealed a list of stem cell‐specific genes in high‐grade astrocytomas, which was likely to have critical roles in determining the “stem” characteristics of high‐grade astrocytomas.

bFGF and heparin but not laminin are necessary factors in the mediums that affect NSCs differentiation into cholinergic neurons

Abstract Objectives: Neural stem cells (NSCs) are self-renewed, pluripotent cells that can differentiate into neurons, astrocytes and oligodendrocytes. Cholinergic neurons are an important kind of neurons that play an essential role in the treatment of Parkinsonism and epilepsy. We are interested in how different mediums affect NSCs differentiation into cholinergic neurons. Methods: NSCs were isolated from the striatum corpora of embryonic brain in a 14-day pregnant rat. Cells were cultured in basic mediums [F12/DMEM (1:1) including 1% B27 (v/v) and 20 ng/ml EGF] but with different combinations of three supplements: bFGF (20 ng/ml), heparin (5 μg/ml) and laminin (1 μg/ml). After 7 days culturing, cells were immunized with choline acetyltransferase (ChAT), a marker enzyme of cholinergic neuron. Results: We found ChAT could not be detected in the basic mediums with only one supplement. Then, we tested the combination of two out of three. We found that ChAT positive cells could only be detected in the medium with bFGF and heparin (FH). However, when we added the laminin into the FH, more ChAT positive cells appeared. Discussion: This finding suggests that bFGF and heparin are essential in the mediums that affect NSCs differentiation into cholinergic neurons, and laminin is an important positive factor in this process.

Knockdown of PKCε expression inhibits growth, induces apoptosis and decreases invasiveness of human glioma cells partially through Stat3.

Glioma is the most common primary central nervous system tumor. Despite considerable research effort, little progress has been made in the therapeutic treatment of this disease. Protein kinase Cε (PKCε), an important intracellular signaling molecule, modulates diverse cellular functions, including cell proliferation, apoptosis, invasion and differentiation. The aim of the study is to investigate whether knockdown of PKCε expression by RNA interference (RNAi) could affect the growth, apoptosis and invasion of human glioma cells, and the involvement of the signal transducer and activator of transcription 3 (Stat3) signaling pathway in these effects. Our data showed that knockdown of PKCε expression inhibited proliferation, induced apoptosis and decreased invasiveness of human glioma cell lines U251 and U87, as well as suppressed the growth of U87 cell-derived tumors in nude mice. Moreover, PKCε physically interacts with Stat3, and knockdown of PKCε expression attenuated Stat3Ser727 phosphorylation and B-cell lymphoma-extra large (Bcl-xL) expression in the two human glioma cell lines. These results support an important role for PKCε in glioma cell growth, apoptosis and invasion, and PKCε exerting its above effects at least in part through Stat3. Thus, PKCε has the potential to be an attractive therapeutic target for glioma therapy.

Mammalian diaphanous-related formin 1 is required for motility and invadopodia formation in human U87 glioblastoma cells

Characterized by invasive growth and infiltrative dissemination, glioma is poorly diagnosed and prognosed at present. The mammalian diaphanous-related formin 1 (mDRF1), which is involved in a number of actin-related biological processes, has been found to participate in the process of invasion and metastasis in human breast cancer cells and to show abnormal expression under pathological conditions. However, the role of mDRF1 in glioma is not clear. In this study, we carried out a comprehensive analysis of the effects of mDRF1 on human glioma. We used siRNA to knock down mDRF1 expression in highly invasive U87 malignant glioma (MG) cells and examined the changes in cell proliferation, apoptosis, invasion and migration. Atomic force microscopy was used to examine invadopodia formation. Immunohistochemical and immunocytochemical assays were used to analyze the cellular localization and the expression levels of mDRF1 in human glioma tissue and in the U87 MG cells. Following the transfection of U87 MG cells with siRNA-mDRF1, their in vitro proliferation was significantly decreased, apoptosis was markedly increased, and invasion and metastasis were significantly inhibited. The results from atomic force microscopy revealed that invadopodia were formed at leading the edge of the U87 MG cells. However, following the silencing of mDRF1 by siRNA, the edge of the cells became smooth and the invadopodia disappeared. For in vivo experiments, nude mice were transplanted with tumor cells and then treated with siRNA-mDRF1. The results revealed that treatment with siRNA-mDRF1 significantly inhibited tumor growth and led to a decrease in the weight of the transplanted tumor. In conclusion, our data demonstrate that mDRF1 is highly expressed in human glioma tissue. The knockdown of mDRF1 in U87 MG cells led to a sharp decline in their invasive and metastatic ability, which effectively reduced the spread of glioma cells into the surrounding areas. To our knowledge, this is the first report showing that mDRF1 is a promising target for the treatment of malignant gliomas.