Silencing of PTK7 in Colon Cancer Cells: Caspase-10-Dependent Apoptosis via Mitochondrial Pathway

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers

A Surface Energy Transfer Nanoruler for Measuring Binding Site Distances on Live Cell Surfaces

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Forster resonance energy transfer (FRET) have limitations due to short detection distances and strict oriental ions. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the firs: time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.Chinese Government ; Academic Women Madelyn Lockhart Dissertation Fellowship ; NIH ; NSF ; National Basic Research Program of China [2007CB935603, 2010CB732402]; [2009ZX10004-312

Cell viability after incubation with caspase inhibitors prior to transfaction of PTK7 siRNA.

<p>(A) Apoptosis induced by knocking down PTK7 was caspase-dependent. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (B) Caspase-10 inhibitor totally blocked the apoptosis induced by knock down of PTK7. Data: mean±s.d. of three independent experiments, *Student's t-test: P<0.05.</p

Involvement of mitochondrial pathway in apoptosis induced by PTK7 scilencing.

<p>(A) Fluorescence microscope detection of mitochondrial membrane potential in treated HCT 116 cells. (B) Flow cytometry detection of mitochondrial membrane potential in treated HCT 116 cells. (C) Activation of caspase-9 involved in apoptosis induced by knocking down PTK7. The membrane was stripped and reprobed by β-actin antibody, as a loading control.</p

Mitochondria and caspase-10 involvement in the apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.

<p>(A) Fluorescence microscope detection of mitochondrial membrane potential in treated p53-null HCT 116 cells. (B) Flow cytometry detection of mitochondrial membrane potential in treated p53-null HCT 116 cells. (C) Cell viability after incubation with caspase inhibitors prior to transfaction of PTK7 siRNA. Caspase-10 inhibitor totally blocked the apoptosis induced by knock down of PTK7. Data: mean±s.d. of three independent experiments, *Student's t-test: P<0.05.</p

The activation of caspase-10 in apoptosis induced by knocking down of PTK7.

<p>(A) Western blot analysis of procaspase-10 and Bid in HCT 116 cells transfected by PTK7 siRNAs. The membrane was stripped and reprobed by β-actin antibody, as a loading control. (B) Caspase-10 activity in HCT 116 cells: untreated and treated with vehicle, nonspecific siRNA and siRNA. Results were given as ratios to caspase-10 activity in untreated cells. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05.</p

PTK7 expression and cell apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.

<p>(A) Flow cytometry assay for the binding of the PE-labeled anti-PTK7 with p53-null HCT 116 cells (Grey curves). The black curves represent the background binding of anti-IgG-PE. The concentration of the antibody in the binding buffer was 2 µg/µL. (B) The number of live p53-null HCT 116 cells was counted on day 4 after treatment with vehicle, nonspecific siRNA and PTK7 siRNA. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (C) BrdU incorporation relative to untreated cells detected by flow cytometry. p53-null HCT 116 Cells were incubated with 10 µM BrdU for 2 h after 48 h of treatment. The amount of BrdU incorporation was normalized to the untreated group. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (D) Apoptosis occurrence in p53-null HCT 116 cells detected by Annexin V/PI stain on days 4 after transfection. Cells stained negative for both Annexin V and PI were considered healthy and percentage was shown in the figure.</p

PTK7 expression in HCT 116 cells after treatment with vehicle, nonspecific siRNA and PTK7 siRNA.

<p>(A) Flow cytometry assay for the binding of the PE-labeled anti-PTK7 with HCT 116 cells (Grey curves). The black curves represent the background binding of anti-IgG-PE. The concentration of the antibody in the binding buffer was 2 µg/µL. (B) Western blot analysis of PTK7 in HCT 116 cells transfected by PTK7 siRNAs. The membrane was stripped and reprobed by β-actin antibody as a loading control. (C) Suppression of PTK7 mRNA expression in HCT 116 cells by PTK7 siRNAs. Cells were harvested after 48 h of treatment. RT-PCR was performed using gene-specific primers. The amount of PTK7 mRNA expression was normalized to the untreated group. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05.</p