OBJECTIVETo explore the relationship between gamma-synuclein gene expression and CpG island demethylation in colorectal cancer(CRC), and the relationship between the demethylation and clinicopathological factors of CRC.

METHODSThe expression of gamma-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues(NNAT) by RT-PCR. CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine(5-aza-C). Before and after the treatment, the expression of gamma-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of gamma-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylation-specific PCR(real-time MSP). The relationship between the demethylation of gamma-synuclein in CRC and clinicopathological factors was analyzed.

RESULTSThe mean gamma-synuclein mRNA expression was 0.66+/-0.34 in CRC samples, which was much higher than 0.45+/-0.26 in NNAT samples(P=0.011). 5-aza-C could induce expression and demethylation of gamma-synuclein in COLO205, LoVo and SW480 cells. gamma-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples. The demethylated status of gamma-synuclein was much higher in CRC samples than that in NNAT samples(P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC(P<0.05).

CONCLUSIONThe upregulation of gamma-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.

Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; CpG Islands ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Humans ; Prognosis ; RNA, Messenger ; genetics ; gamma-Synuclein ; genetics ; metabolism

OBJECTIVETo construct &gamma;-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.

METHODSTotal RNA was extracted from colon cancer cell line HT29 and the cDNA of &gamma;-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.

RESULTSHuman &gamma;-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-&gamma;-synuclein protein in vitro. &gamma;-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).

CONCLUSIONExpression of &gamma;-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Cell Adhesion ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; gamma-Synuclein ; genetics

OBJECTIVESTo evaluate the expression of synuclein-&gamma; (SNCG) and metalloproteinase 9 (MMP-9) both in the invasive ductal breast cancer samples and T47D and T47D(SNCG)- cell lines, to investigate the correlation between SNCG and MMP-9.

METHODSTotally 96 invasive ductal breast cancer samples (female, mean age of (56 ± 8) years) were collected between June 2009 and June 2012. The expressions of SNCG and MMP-9 were investigated by immunohistochemistry. T47D and SNCG knock down T47D(SNCG)- cell lines were established and SNCG and MMP-9 protein expression were investigated by Western blot and gene expression by real-time PCR.

RESULTSAmong 96 samples, 26 (27.1%) of them co-expressed SNCG and MMP-9, 30(31.2%) of them expressed neither SNCG nor MMP-9. The expression of SNCG was correlated with the expression of MMP-9 (r = 0.655, P = 0.000).SNCG mRNA level of T47D cell line was 13.5 fold of T47D(SNCG)- cell line and SNCG protein expression was 2.1 fold. While MMP-9 mRNA level of T47D cell line was 7.3 fold of T47D(SNCG)- cell line and MMP-9 protien expression was 1.6 fold.When SNCG was knocked down, the expression of MMP-9 decreased.

CONCLUSIONSSNCG and MMP-9 are significantly correlated with each other in breast cancer. SNCG may promote the invasion and metastasis of breast cancer mediated by up-regulating the expression of MMP-9.

Aged ; Breast Neoplasms ; metabolism ; Female ; Gene Expression ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; gamma-Synuclein ; metabolism

OBJECTIVETo investigate the expression of SNCG in colorectal cancer with liver metastasis and its clinical significance.

METHODSSurgical specimens were collected from 217 colorectal cancer patients with complete clinical and follow up data between January 1999 and December 2003. There were 113 cases with liver metastasis and 104 without liver metastasis. SNCG expression was identified by immunohistochemistry. Association of SNCG expression with clinicopathologic factors and prognosis of colorectal cancer was accessed.

RESULTSThe positive rate of SNCG in colorectal cancer with and without liver metastasis was 68.1% and 27.9%, respectively, and the difference was statistically significant(P<0.05). Logistic regression analysis showed that SNCG expression was an independent factor associated with the presence of liver metastasis(OR=8.29, 95%CI: 3.37-20.37, P<0.01). In synchronous colorectal liver metastasis, the median survival time of SNCG-negative and SNCG-positive was 12.6 months and 8.2 months, respectively(Log Rank, P<0.05). Multivariate Cox analysis showed that SNCG expression was an independent prognostic factor for colorectal cancer with synchronous liver metastasis(RR=1.97, 95%CI:1.10-3.53, P<0.05).

CONCLUSIONSHigh expression of SNCG is present in the tumor tissue in patients with liver metastasis from colorectal cancer. SNCG may be used as a predictive biomarker for colorectal liver metastases and is an important prognostic factor in patients with liver metastasis from colorectal cancer.

Colorectal Neoplasms ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Liver Neoplasms ; secondary ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Prognosis ; Retrospective Studies ; gamma-Synuclein ; metabolism

OBJECTIVETo observe the sensitivity of the PC-3 cell lines transfected with the PCI-NEO-SNCG plasmid to Cisplatin (DDP), 5-Fluorouracil (5-FU), Adriamycin (ADM), Vincristine (VCR) and Paclitaxel (TAX), and to explore the influence of the SNCG expression on the effectiveness of anti-tumor drugs.

METHODSThe plasmids PCI-NEO and PCI-NEO-SNCG were transfected into the hormone-independent prostate cancer cell lines PC-3. RT-PCR was adopted to examine the expression of SNCG in the PC-3 cell lines. The MTT method was employed to detect the suppressive effects of different anti-tumor drugs (DDP, ADM, 5-FU, VCR and TAX) on the cell lines transfected with PCI-NEO and PCI-NEO-SNCG. Flow cytometry was used to analyze the cell cycles and apoptosis of the transfected cells treated with TAX.

RESULTSThe 5 anti-tumor drugs suppressed the growth of the cell lines transfected with the plasmids PCI-NEO and PCI-NEO-SNCG in a time-dependant manner. The comparison between the growth-suppressing effects of different anti-tumor drugs on the PC-3 cell lines showed no significant differences between the group transfected with PCI-NEO and that with PCI-NEO-SNCG in DDP, 5-FU, ADM and VCR (P > 0.05), while the rate of suppression of TAX on the latter cell lines was significantly lower than that on the former (P < 0.01). Compared with the PCI-NEO-SNCG plasmid transfected cell lines, after treated with TAX for 48 hours, those transfected with the PCI-NEO plasmid exhibited a significantly larger proportion of cells remaining in the G2-M stage (P < 0.01), a smaller proportion in the G0-G1 and S stages (P < 0.01) and a significantly higher expression of Caspase-3 (P < 0.01).

CONCLUSIONThe significant reduction of the growth-suppressing effect of TAX in the SNCG-transfected PC-3 cell lines suggests that the expression of SNCG may restrain the effect of TAX. These findings have provided evidence and guide to the individual chemotherapy of prostate cancer.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Drug Screening Assays, Antitumor ; Humans ; Male ; Neoplasm Proteins ; genetics ; Paclitaxel ; pharmacology ; Prostatic Neoplasms ; Transfection ; gamma-Synuclein ; genetics

PURPOSE: Synuclein has been identified as an important neuroprotein for developing pathologic deposits in Alzheimer's and Parkinson's disease patients. gamma-synuclein is also known as a breast cancer-specific gene 1 thats's not found in normal breast tissues but it has been reported to be overexpressed in breast cancer, ovarian cancer and other tumors. To evaluate the availability of gamma-synuclein expression as a prognostic factor for infiltrative breast cancer, we analyzed its correlation with the clinical parameters and the HER-2/neu gene expression. METHODS: Two hundred fiffty samples of breast cancer tissues embedded in paraffin and that were obtained from the infiltrative breast cancer patients who were operated in our institution from January 1995 to December 2000 were analyzed with employing the tissue microarray technique. The expression of gamma-synuclein was studied with immunohistochemistry and with using gamma-synulcein antibodies. One hundred thirty one cases that showed favorable staining were selected and studied retrospectively. RESULTS: Fiffty five% (71/131) of the patients showed gamma-synuclein overexpression. The histopathological findings that significantly correlated with gamma-synuclein overexpression were the number of metastatic lymph nodes (p<0.01) and the cancer stage (p<0.01). Using the same tissue mircoarray, the HER-2/neu gene expression and gamma-synuclein expression also showed statistically significant correlation (p=0.04). CONCLUSION: gamma-synuclein overexpression showed significant correaltion with lymph node metastasis and cancer stage. It also showed significant relevance with the HER-2/neu gene expression, and that is already known to be a prognostic factor for breast cancer. Therefore gamma-synuclein may be a useful prognostic factor for infiltrative breast cancer and further studies on the its correlation with survival, local recurrence, and distant metastasis should be conducted
Antibodies ; Breast Neoplasms* ; Breast* ; gamma-Synuclein* ; Gene Expression ; Humans ; Immunohistochemistry ; Lymph Nodes ; Neoplasm Metastasis ; Ovarian Neoplasms ; Paraffin ; Parkinson Disease ; Recurrence ; Retrospective Studies ; Synucleins

OBJECTIVETo evaluate the relationship between idiopathic Parkinson's disease (PD) and two polymorphisms (C243G and A377T) of the gamma-synuclein gene in a Chinese Han population of Shanghai area.

METHODSPolymorphic genotyping was performed with PCR-RPLP technique. Association analysis was carried out in 145 unrelated idiopathic PD patients and 184 age-matched healthy controls.

RESULTSThe authors failed to detect any distributional difference of the C243G and A377T polymorphisms of the gamma-synuclein gene between PD cases and control subjects, nor did they find any association.

CONCLUSIONThese data do not support that gamma-synuclein gene C243G and A377T polymorphisms are involved in idiopathic PD onset in the Han population of Shanghai area.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Gene Frequency ; Genotype ; Humans ; Middle Aged ; Nerve Tissue Proteins ; genetics ; Parkinson Disease ; genetics ; Polymorphism, Single Nucleotide ; Synucleins ; gamma-Synuclein

The synuclein family consists of three distinct genes, alpha-synuclein, beta-synuclein, and gamma-synuclein. The alpha-synuclein and beta-synuclein are predominately expressed in brain and especially alpha-synuclein is related with Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies. The gamma-synuclein was first identified as breast cancer specific gene 1. It is expressed in the peripheral nervous system and also detected in breast and ovarian cancers. The gamma-synuclein is also known to mediate metastasis of breast and ovarian cancer cells. Insulin-like growth factor 1 (IGF-I) is one of the growth factors that plays an important role in cell proliferation and migration in cancer cells, as well as in normal cells. In this study, we investigated the migrations of SKOV-3, MDAMB-231, and HeLa cells by the recombinant synuclein proteins (alpha-, beta-, and gamma-synucleins) and IGF-I and the molecular mechanism. Furthermore, we investigated the membrane ruffle formation of SKOV-3 cells by recombinant synuclein proteins and IGF-I. As a result, synucleins and IGF-I were found to induce cancer cell migrations. Simultaneous synucleins and IGF-I treatment on the cancer cells induced more migrations than the individual synuclein or IGF-I treatments. The synucleins or IGF-I treatments increased the expressions of membrane-type1 matrix metalloproteinase (MT1-MMP) and cluster of differentiation 44 (CD44). Moreover, simultaneous synucleins and IGF-I treatments further increased the expressions of MT1-MMP and CD44. The synucleins and IGF-I promoted the conformational change of actin filaments, and then this led to the membrane ruffle formation.
Actin Cytoskeleton ; alpha-Synuclein ; Alzheimer Disease ; beta-Synuclein ; Brain ; Breast ; Breast Neoplasms ; Cell Movement ; Cell Proliferation ; Dementia ; gamma-Synuclein ; HeLa Cells ; Humans ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins ; Lewy Bodies ; Matrix Metalloproteinase 14 ; Membranes ; Neoplasm Metastasis ; Ovarian Neoplasms ; Parkinson Disease ; Peripheral Nervous System ; Proteins ; Synucleins

Hsc70-SNCG fusion protein cDNA fragment containing signal peptide sequence of Igkappa, MMP9, and P37 was inserted into the vector pVAX1 to construct recombinant plasmid pVAX-Igkappa-Hsc70-SNCG, pVAX-MMP9-Hsc70-SNCG, and pVAX-P37-Hsc70-SNCG. Three eukaryotic vectors were constructed and verified by restriction enzyme digestion and sequencing. After transfection with recombination plasmids in QM-7 cells, the transient expression and secretion of three fusion proteins were detected by ELISA and Western Blot. The results suggested that Hsc70-SNCG carrying three different signal peptides could be expressed and secreted by transfected cells, and three signal peptides effectively directed secretion of fusion protein by QM-7 cells. BALB/c mice were immunized by three plasmids using gene gun system. The serum levels of anti-SNCG antibodies in mice were measured by ELISA. The results showed that three secreted plasmids could stimulate humoral immune responses to SNCG in mice, which depended on the secreted expression levels induced by signal peptides.
Animals ; Genetic Vectors ; Humans ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Neoplasm Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; gamma-Synuclein ; biosynthesis ; genetics ; immunology

OBJECTIVETo detect breast cancer specific gene 1 (BCSG1) expression in different breast tissue, analysis its correlation with clinical parameters and evaluate the prognosis of breast cancer.

METHODSThe expression of BCSG1 was detected by reverse transcription-polymerase chain reaction (RT-PCR) in surgical specimens from 84 cases of breast disease patients selected randomly at XinHua Hospital affiliated with Shanghai Second Medical University from September 1999 to December 2002. Of 84 cases, 72 case were breast cancer. Statistic analysis BCSG1 gene expression correlation with clinical parameters of breast cancer. 72 breast cancers were followed up (4 - 43 months) to set up independent prognosis factor by survival analysis.

RESULTSBCSG1 was undetectable in all benign breast lesions, while was detectable in 36.1% of all breast cancer samples (26/72), in which 79.2% of stage III/IV cases were positive (19/24). The expression of BCSG1 was tightly correlated with the stage (P = 0.000) and the size of tumor (P = 0.007). Both ER (P = 0.027) and BCSG1 (P = 0.001) were the independent prognosis factor of breast cancer.

CONCLUSIONBCSG1 is one of independent tumor marker of breast cancer, the expression of BCSG1 is closely correlated to the stage of breast cancer and the tumor size. Maybe, BCSG1 is a new prognosis factor of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; diagnosis ; genetics ; pathology ; Female ; Gene Expression ; Humans ; Middle Aged ; Neoplasm Proteins ; genetics ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; gamma-Synuclein ; genetics