Silencing of ENO1 inhibits the proliferation, migration and invasion of human breast cancer cells.

PURPOSE: Studies have shown that α-enolase ENO1 is involved in the regulation of cancer cell proliferation and metastasis. However, the role of ENO1 is yet to be explored in breast cancer. This study was undertaken to explore the role and therapeutic potential of ENO1 in breast cancer. METHODS: Expression analysis was carried out by qRT-PCR. Transfections were performed by Lipofectamine 2000 reagent. WST-1 assay was used for cell viability. Wound healing assay was used for cell migration analysis. Western blot analysis was used to determine protein expression. RESULTS: The results showed that the expression of ENO1 was significantly upregulated in breast cancer by up to 4-fold. Silencing of ENO1 caused significant decline in the proliferation rate and colony formation of the SK-BR-3 breast cancer cells. The decrease in the proliferation rate of the ENO1 cells was due to the induction of apoptosis as revealed by DAPI staining. Annexin V/propidium iodide (PI) showed a significant increase in the apoptotic SK-BR-3 cells. The apoptosis percentage was 2.17 in si-NC and 23.1% in si-ENO1 transfected SK-BR-3 cells. The apoptosis induction was also accompanied by increase in Bax and decrease in Bcl-2 expression. ENO1 silencing also resulted in the arrest of the SK-BR-3 cells in the G2/M phase of the cell cycle which was also associated with depletion of Cdc2, Cdc25 and cyclin B1 expression levels. ENO1 silencing also caused decrease in the migration and invasion of the SK-BR-3 cells as revealed by the wound healing and transwell assays. CONCLUSION: These findings suggest that ENO1 has oncogenic properties in breast cancer which can be exploited in breast cancer treatment.

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.

Crocodilian tau-crystallin: overexpression, purification, and characterization.

tau-Crystallin is a taxon-restricted crystallin found in eye lenses of reptiles and a few avian species but presumably absent in mammals. The level of tau-crystallin in the lens varies among different species. In the crocodile lens, it is the least abundant crystallin and is present in trace amounts. We present a method for cloning, overexpression, and purification of crocodilian tau-crystallin utilizing a combination of gel filtration and ion-exchange chromatography yielding an extremely purified protein. The protein gets profusely expressed resulting in a fairly high yield and exists as a monomeric entity of 47.5 kDa molecular mass. The recombinant tau-crystallin exists in a properly folded native state as probed by circular dichroism and fluorescence spectroscopy and exhibits enolase activity.

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris.

tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.