[Fat1 inhibits cell proliferation via ERK signaling pathway in esophageal squamous cell carcinoma].

Objective: To clarify the mechanism of Fat1 on the proliferation of esophageal squamous cell carcinoma (ESCC). Methods: KYSE450 cells were transfected with Plko.1-puro-GFP-shRNA-Fat1 plasmid and real time polymerase chain reaction (PCR) was used to verify the efficiency of Fat1 knockdown. The effects of Fat1 and extracellular regulated protein kinase (ERK) pathway inhibitor U0126 on the proliferation of ESCC cells were detected by methyl thiazolyl tetrazolium (MTT). Colony formation assay was used to detect the colony formation ability. Cell cycle was detected by live cell imaging. Western blot was used to observe the level of target protein. Mouse xenograft assay was applied to detect the effect of Fat1 knockdown on KYSE450 cell tumor growth. Immunohistochemistry was used to detect the expressions of related proteins in tumor sections. Results: The efficiency of Fat1 knockdown was (77.1±6.9)% in Fat1 sh1 group and (77.7±7.1)% in Fat1sh2 group. Compared with the control group, the cell proliferation and the expression of p-ERK1/2 were significantly increased in Fat1 sh1 and Fat1sh2 group (P<0.05). After U0126 treatment, the effect of Fat1 knockdown on the proliferation of KYSE450 cells disappeared, and the expression of p-ERK1/2 in KYSE450 cells decreased to a level similar to that in the control group. The number of cell clones in the control group was (72±8), lower than (155±28) and (193±9) in the Fat1sh1 and Fat1sh2 groups, respectively (P<0.05). In KYSE450 cell, division time was shortened from 1 622±32 min in control group to 1 408±29 min in Fat1 sh1 group, the difference was statistically significant (P<0.05). Compared with the control group, the tumor volume of Fat1 knockdown group increased significantly. The tumor weight of control group and Fat1 knockdown group were (0.224±0.028) g and (1.532±0.196) g, respectively, at 4 weeks after inoculation, and the difference was statistically significant (P<0.05). Conclusion: Fat1 inhibits cell proliferation via ERK signaling in ESCC.

The effect of curcumin on cell adhesion of human esophageal cancer cell.

OBJECTIVE: Esophageal cancer is the 8th most common cancers worldwide and the 6th most common cause of death among cancers. Curcumin has been reported to have the function of anti-inflammatory, antioxidant, anti-rheumatoid, and anti-atherosclerosis role. It can also reduce lipid, eliminate free radicals and inhibit the growth of the tumor. Many reports had suggested that curcumin has shown great potential in the treatment of tumors by inducing apoptosis. Little is known about the effects of curcumin on cell adhesion of tumor cancer. Therefore, in this study, we attempted to look for a new approach to target resistant cells and improve efficacy without toxicity. MATERIALS AND METHODS: Human esophageal cancer cell line (Eca-109 cells) was cultured. Cell adhesion was detected under a microplate reader. Reactive oxygen species were measured using Fluostar Omega Spectrofluorimeter. SOD activity and GSH content in cells were detected by commercial determination kit. The expression of p-JAK, p-STAT3 and STAT3 were measured by Western blot and RT-PCR. RESULTS: Cell adhesion assay showed curcumin enhances cell-cell adhesion and cell-matrix adhesion in Eca-109 cells. ROS levels, SOD activity and total GSH content were detected and the results showed curcumin decreases intracellular ROS levels but increases SOD activity and total GSH content. Then, NAC (ROS inhibitor) and ICI (ER inhibitor) were pre-treated. Results showed ICI reversed the decreasing of intracellular ROS levels and the increasing of SOD activity and total GSH content affected by curcumin, but NAC had no such impact. Taken together, ER rather than ROS involves in cell adhesion affected by curcumin. Meanwhile, the downregulating of p-JAK, p-SATA3 and total STAT3 were caused by curcumin but NAC had no such influence. They were reversed by ICI, but NAC had no such influence. CONCLUSIONS: Curcumin could increase cell adhesion through inhibiting JAK/STAT3 mediated by ER in Eca-109.

Gene expression profiling of rat testis development during the early post-natal stages.

Spermatogenesis is a complex biological process that requires precise regulation of gene expression in the germ cells and their surrounding somatic cells. Some testis-specific genes are involved in different stages of spermatogenesis; however, the precise mechanisms of stage-specific spermatogenesis are still not elucidated. In this study, we first examined the expression patterns of SYCP3, Tnp2, CDH1, glial cell-line-derived neurotropic factor (GDNF) and GFRA1 mRNAs on post-natal days (PNDs) 2, 4, 6, 8, 10, 12, 15, 20, 25 and 30 in rat testis. SYCP3 mRNA was firstly detected from PND 15, while Tnp2 transcript was only found on PND 30. CDH1 mRNA was highly expressed before PND 6, but decreased dramatically on PND 8, then gradually increased until it started to decrease after 12 dpp. Low GDNF and GFRA1 mRNAs were found before PND 6, but gradually increased to the peak on PND 12, then gradually decreased to low level. According to the expression patterns of CDH1, GDNF and GFRA1, we hypothesized that PNDs 6-10 are critical period in the early spermatogenesis. We, therefore, explored gene expression pattern on PNDs 6, 8 and 10 using cDNA microarray. 700 (PND 8 vs PND 6), 4519 (PND 10 vs PND 8), and 4298 (PND 10 vs PND 6) differentially expressed genes (≥ 2-fold) were identified from the comparisons, which cover thousands of gene ontology categories (GO terms) and hundreds of signalling pathways. High consistency between microarray data and quantative real-time PCR (qRT-PCR) was verified from five genes (LOC686076, Trib3, Cxcl6, LOC682508 and C2cd4d). These data provide more information to understand the precisely regulatory mechanism at the early stage of spermatogenesis.

Characterization of pco-1, a newly identified gene which regulates purine catabolism in Neurospora.

A new gene of Neurospora crassa, designated pco-1, was characterized and shown to regulate the expression of several genes which encode enzymes required for the catabolism of purines. Unlike the wild type, a pco-1 mutant created by repeat-induced point mutation cannot utilize purines as a nitrogen source. The PCO1 protein contains a Zn(II)2Cys6 binuclear cluster motif near its N-terminus, followed by a putative coiled-coil motif. A chemical crosslinking experiment demonstrated that PCO1 forms homodimers. PCO1 binds to CGG-N6-CCG elements located in the upstream promoter region of four genes encoding purine catabolic enzymes. Northern blot analysis demonstrated that a functional PCO1 protein is required for induction of xdh, which encodes xanthine dehydrogenase. Moreover, PCO1 was required for induction of three different purine catabolic enzymes. Two glutamine-rich domains occur in the C-terminal region of PCO1 and at least one of the glutamine-rich regions is required for PCO1 function, suggesting that they might play a role in transcriptional activation. The PCO1 protein does not interact with the global-acting NIT2 protein or the negative-acting NMR protein that functions in nitrogen catabolite repression. Induction of the xdh gene and synthesis of xanthine dehydrogenase is completely dependent upon PCO1, but does not require the global-acting NIT2 protein, suggesting that it is controlled by a novel regulatory mechanism.