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DNA aptamer evolved by cell-SELEX for recognition of prostate cancer.

Wang Y, Luo Y, Bing T, Chen Z, Lu M, Zhang N, Shangguan D, Gao X - PLoS ONE (2014)

Bottom Line: Wy-5a shows high specificity to the target cells with dissociation constants in the nanomolar range, and does not recognize other tested PCa cell lines and other tested tumor cell lines.The staining of clinical tissue sections with fluorescent dye labeled Wy-5a shows that sections from high risk group with metastasis exhibited stronger fluorescence and sections from Benign Prostatic Hyperplasia (BPH) did not exhibit notable fluorescence, which suggests that aptamer Wy-5a may bind to protein related to the progression of PCa.The high affinity and specificity of Wy-5a makes this aptamer hold potential for application in diagnosis and target therapy of PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, the third affiliated hospital of Sun Yat-sen University, Guangzhou, Guangdong, China.

ABSTRACT
Morbidity and mortality of prostate cancer (PCa) have increased in recent years worldwide. Currently existing methods for diagnosis and treatment do not make the situation improve, especially for hormone refractory prostate cancer (HRPC). The lack of molecular probes for PCa hindered the early diagnosis of metastasis and accurate staging for PCa. In this work, we have developed a new aptamer probe Wy-5a against PCa cell line PC-3 by cell-SELEX technique. Wy-5a shows high specificity to the target cells with dissociation constants in the nanomolar range, and does not recognize other tested PCa cell lines and other tested tumor cell lines. The staining of clinical tissue sections with fluorescent dye labeled Wy-5a shows that sections from high risk group with metastasis exhibited stronger fluorescence and sections from Benign Prostatic Hyperplasia (BPH) did not exhibit notable fluorescence, which suggests that aptamer Wy-5a may bind to protein related to the progression of PCa. The high affinity and specificity of Wy-5a makes this aptamer hold potential for application in diagnosis and target therapy of PCa.

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Related in: MedlinePlus

Cell-specific internalization of aptamer Wy-5a assessed by flow cytometry assay (left) and confocal imaging (right).W/O treatment: Cells were incubated with Wy-5a; Pretreatment: cells were pretreated by trypsin and then incubated Wy-5a; aftertreatment: cells were incubated Wy-5a and then treated by trypsin; Lib: negtive control, cells were incubated with FITC-labeled DNA library; blank: is the background fluorescence of untreated cells.
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pone-0100243-g006: Cell-specific internalization of aptamer Wy-5a assessed by flow cytometry assay (left) and confocal imaging (right).W/O treatment: Cells were incubated with Wy-5a; Pretreatment: cells were pretreated by trypsin and then incubated Wy-5a; aftertreatment: cells were incubated Wy-5a and then treated by trypsin; Lib: negtive control, cells were incubated with FITC-labeled DNA library; blank: is the background fluorescence of untreated cells.

Mentions: Previous studies have shown that oligonucleotides longer than 25 bases could not freely penetrate the membrane [33], [34]. Wy-5a is a 52-base oligonucleotide, it targets a membrane protein. In order to investigate whether Wy-5a can internalize into cells through receptor-mediated endocytosis, PC-3 cells were incubated with Wy-5a at 37°C for 2 h. The increase of tempreture from 4°C to 37°C did not much affect the binding of Wy-5a to PC-3 cells (Figure S2). The flow cytometry assay (Figure 6) showed that pre-treatment of cells with trypsin caused almost complete loss of aptamer binding (compared with unbound DNA labrary). However, treatment of cell with trypsin after aptamer binding only caused partial loss of aptamer binding, suggesting that some Wy-5a might internalize into cells. In the confocal image of PC-3 cell after incubation with Wy-5a at 37°C for 2 h (Figure 6), strong fluorescence signal was observed on cell membrane and weak fluorescence signal was found in whole cells. But the the confocal image of cells icubated DNA library did not show significant fluorescence signal except for some nonspecific bright spots dispersedly adsorbed on cells. These results suggest that Wy-5a may be involved in receptor-mediated endocytosis.


DNA aptamer evolved by cell-SELEX for recognition of prostate cancer.

Wang Y, Luo Y, Bing T, Chen Z, Lu M, Zhang N, Shangguan D, Gao X - PLoS ONE (2014)

Cell-specific internalization of aptamer Wy-5a assessed by flow cytometry assay (left) and confocal imaging (right).W/O treatment: Cells were incubated with Wy-5a; Pretreatment: cells were pretreated by trypsin and then incubated Wy-5a; aftertreatment: cells were incubated Wy-5a and then treated by trypsin; Lib: negtive control, cells were incubated with FITC-labeled DNA library; blank: is the background fluorescence of untreated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067300&req=5

pone-0100243-g006: Cell-specific internalization of aptamer Wy-5a assessed by flow cytometry assay (left) and confocal imaging (right).W/O treatment: Cells were incubated with Wy-5a; Pretreatment: cells were pretreated by trypsin and then incubated Wy-5a; aftertreatment: cells were incubated Wy-5a and then treated by trypsin; Lib: negtive control, cells were incubated with FITC-labeled DNA library; blank: is the background fluorescence of untreated cells.
Mentions: Previous studies have shown that oligonucleotides longer than 25 bases could not freely penetrate the membrane [33], [34]. Wy-5a is a 52-base oligonucleotide, it targets a membrane protein. In order to investigate whether Wy-5a can internalize into cells through receptor-mediated endocytosis, PC-3 cells were incubated with Wy-5a at 37°C for 2 h. The increase of tempreture from 4°C to 37°C did not much affect the binding of Wy-5a to PC-3 cells (Figure S2). The flow cytometry assay (Figure 6) showed that pre-treatment of cells with trypsin caused almost complete loss of aptamer binding (compared with unbound DNA labrary). However, treatment of cell with trypsin after aptamer binding only caused partial loss of aptamer binding, suggesting that some Wy-5a might internalize into cells. In the confocal image of PC-3 cell after incubation with Wy-5a at 37°C for 2 h (Figure 6), strong fluorescence signal was observed on cell membrane and weak fluorescence signal was found in whole cells. But the the confocal image of cells icubated DNA library did not show significant fluorescence signal except for some nonspecific bright spots dispersedly adsorbed on cells. These results suggest that Wy-5a may be involved in receptor-mediated endocytosis.

Bottom Line: Wy-5a shows high specificity to the target cells with dissociation constants in the nanomolar range, and does not recognize other tested PCa cell lines and other tested tumor cell lines.The staining of clinical tissue sections with fluorescent dye labeled Wy-5a shows that sections from high risk group with metastasis exhibited stronger fluorescence and sections from Benign Prostatic Hyperplasia (BPH) did not exhibit notable fluorescence, which suggests that aptamer Wy-5a may bind to protein related to the progression of PCa.The high affinity and specificity of Wy-5a makes this aptamer hold potential for application in diagnosis and target therapy of PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, the third affiliated hospital of Sun Yat-sen University, Guangzhou, Guangdong, China.

ABSTRACT
Morbidity and mortality of prostate cancer (PCa) have increased in recent years worldwide. Currently existing methods for diagnosis and treatment do not make the situation improve, especially for hormone refractory prostate cancer (HRPC). The lack of molecular probes for PCa hindered the early diagnosis of metastasis and accurate staging for PCa. In this work, we have developed a new aptamer probe Wy-5a against PCa cell line PC-3 by cell-SELEX technique. Wy-5a shows high specificity to the target cells with dissociation constants in the nanomolar range, and does not recognize other tested PCa cell lines and other tested tumor cell lines. The staining of clinical tissue sections with fluorescent dye labeled Wy-5a shows that sections from high risk group with metastasis exhibited stronger fluorescence and sections from Benign Prostatic Hyperplasia (BPH) did not exhibit notable fluorescence, which suggests that aptamer Wy-5a may bind to protein related to the progression of PCa. The high affinity and specificity of Wy-5a makes this aptamer hold potential for application in diagnosis and target therapy of PCa.

Show MeSH
Related in: MedlinePlus