Inhibition of PTK7 expression by PTK7 siRNA
Expression of PTK7 in HCT 116, human colon carcinoma cells, was investigated by flow cytometry (, untreated) and Western blot (, 0 h). Comparing the fluorescence signal of PE-labeled anti-PTK7 to PE-labeled anti-mouse IgG clearly shows that PTK7 is expressed in HCT 116 cells.
PTK7 expression in HCT 116 cells after treatment with vehicle, nonspecific siRNA and PTK7 siRNA.
Expression of PTK7 was knocked down using PTK7-targeted siRNA and the flow cytometry results for the targeted cells were compared to those exposed to vehicle only, nonspecific siRNA or untreated, as shown in . After 48 hours, the peak of anti-PTK7-PE in HCT 116 transfected with PTK7 siRNA shifted back to the peak of the background control protein, anti-IgG-PE, indicating that the PTK7 expression level in HCT 116 cells transfected with PTK7 siRNA greatly decreased. At the same time, there was no corresponding shift in the control siRNA or vehicle-treated groups, indicating that neither the HiPerFect transfection reagent nor the nonspecific siRNA affected PTK7 expression. When PTK7 expression was probed after 12 h, 24 h, 30 h and 48 h of transfection using Western blot (), the results clearly showed that the level of PTK7 expression decreased after 48 h of transfection. In addition, total mRNA was extracted from the untreated, vehicle, nonspecific siRNA, and PTK7 siRNA groups. As shown in , PTK7 siRNA induced 75–80% reduction of PTK7 mRNA in HCT 116 cells. These results indicated that both PTK7 protein and mRNA expression levels were greatly decreased by PTK7 siRNA. This proved the function and efficiency of PTK7 siRNA and provided a solid basis for our study of PTK7's functional role.
Viability and proliferation of PTK7 siRNA-treated HCT 116 cells
The effect of PTK7 suppression on the viability of HCT 116 cells was investigated by counting the total number of live cells every day after transfection. As shown in , the number of live HCT 116 cells transfected with PTK7 siRNA was shown to be significantly different from that of untreated groups on day 4. This finding demonstrated a significant inhibition of cell viability in the HCT 116 cells treated with PTK7 siRNA. To confirm that the decrease of cell viability resulted from suppression of PTK7, the same assays were carried out with HCT 116 cells transfected with nonspecific siRNA or treated only with vehicle. The results showed that the PTK7 siRNA-treated sample contained the smallest number of cells. Although vehicle-treated and nonspecific siRNA-treated cells had smaller cell numbers than untreated cells, there were significantly fewer cells in the PTK7 siRNA-treated sample.
Cell viability in HCT 116 cells after treatment with vehicle, nonspecific siRNA and PTK7 siRNA.
To ascertain the effect of suppression of PTK7 on HCT 116 cell proliferation, a BrdU incorporation experiment was performed to measure DNA synthesis. After 48 h of transfection, cells were seeded in 24-well culture plates and were incubated with 10 µM BrdU for 2 h. Cells were then fixed, and BrdU incorporation was detected using a FITC-labeled anti-BrdU antibody (). Silencing of PTK7 significantly inhibited BrdU incorporation in HCT 116 cells, suggesting a direct effect of PTK7 protein on HCT 116 cell proliferation.
Increase of apoptosis of PTK7 siRNA-treated HCT 116 cells
An Annexin V/PI staining experiment was carried out to study the possibility that knocking down PTK7 could affect the apoptosis of HCT 116 cells. Phosphatidylserine (PS) is located in the inner leaflet of the cell membrane in healthy cells. During apoptosis, PS becomes translocated to the outer surface of the cell membrane, and Annexin V/PI assay detects the PS on the outer surface. The results in show that the PTK7 siRNA group showed significant increase in apoptotic cells on day 4 compared with untreated, vehicle, and nonspecific siRNA control groups.
Changes in mitochondrial membrane potential and activation of caspase-9 in HCT 116 cells treated with PTK7 siRNA
To study the mechanism through which knocking down PTK7 induces apoptosis in HCT 116 cells, the effect of knocking down PTK7 on mitochondrial membrane potential (ΔΨm) was determined by fluorescence microscopy () and flow cytometry (). Dye JC-1 was used as an indicator. In healthy cells, polarized mitochondria have a negative charge, which allows JC-1 dye with delocalized positive charge to enter the mitochondrial matrix and accumulate there. When the critical concentration is exceeded, JC-1 forms J-aggregates, and the cells become fluorescent red (FL2). In apoptotic cells, the mitochondrial membrane potential collapses, and JC-1 cannot accumulate within the mitochondria. In these apoptotic cells, JC-1 remains in the cytoplasm in a green fluorescent monomeric form (FL1). After HCT 116 cells were transfected with siRNA or control as described above and incubated for 48 h or 72 h, the decrease of ΔΨm in HCT 116 cells transfected with PTK7 siRNA was observed. After 72 h of transfection, the percentage of cells with polarized mitochondria was 90%, 87% and 88% for cells in the untreated, vehicle and nonspecific siRNA groups, respectively. However, only 35% of total cells in the PTK7 siRNA group had polarized mitochondria. This trend was seen even at 48 h when only 58% cells had polarized mitochondria. These data suggested that mitochondrial dysfunction was involved in the apoptosis induced by PTK7 knockdown.
Involvement of mitochondrial pathway in apoptosis induced by PTK7 scilencing.
A variety of signalling pathways may be involved in apoptosis, and the mitochondria play a major role. Mitochondrial dysfunction causes the release of cytochrome c, which activates caspase-9, in turn fueling apoptosis. Caspase-9 activation was detected by Western blot after HCT 116 cells were transfected with PTK7 siRNA and cultured for 12 h, 24 h, 30 h, and 48 h, respectively. As shown in , caspase-9 was activated and involved in the apoptosis induced by PTK7 knockdown.
Role of caspase-10 in PTK7-knockdown-induced apoptosis
To determine whether caspases mediate the apoptosis induced by knock down of PTK7, cells were pretreated with a pancaspase inhibitor or one of several single-caspase-specific inhibitors. Rescue of the cells from apoptosis would mean that the inhibited caspase was implicated in PTK7-deficient cell death. After pre-incubation of the HCT 116 cells with 20 µM pancaspase-family inhibitor at 37°C for 3 h, the cells were transfected with siRNA for 48 h. After incubation for 48 h, cell viability was tested using Annexin V/PI (). Cells pre-incubated with pancaspase-family inhibitor showed good cell viability (80±13%) after transfection with PTK7 siRNA, within uncertainty of the cell viability of the nonspecific siRNA group (83±7.5%). Meanwhile, cells directly transfected with PTK7 siRNA had significantly lower cell viability (36±12%). Pancaspase-family inhibitor blocked caspase activity and also blocked apoptosis induced by knock down of PTK7, indicating that the apoptosis induced by knock down of PTK7 is caspase-dependent.
Cell viability after incubation with caspase inhibitors prior to transfaction of PTK7 siRNA.
To investigate which caspase plays the critical role in this apoptosis, HCT 116 cells were pre-treated with caspase-9 inhibitor (Z-LEHD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK), caspase-family inhibitor (Z-VAD-FMK), caspase-1 inhibitor (Z-YVAD-FMK), caspase-10 inhibitor (Z-AEVD-FMK), caspase-2 inhibitor (Z-VDVAD-FMK), or DMSO vehicle at 37°C for 3 h, followed by transfection with PTK7 siRNA. After incubation for 48 h, cell viability was tested by Annexin V/PI using flow cytometry. As shown in , inhibition of caspase-10 blocked apoptosis, with 79±3% of cells viable. This meant that caspse-10 may play a critical role in the apoptotic pathway induced by knock down of PTK7.
To confirm the activation of caspase-10 in apoptosis induced by PTK7 knockdown, procaspase-10 protein levels in cell lysates transfected with PTK7 siRNA were examined using Western blot (). Procaspase-10 decreased after 12 h of transfection and increased after 30 h of transfection. Also, as the linkage between the intrinsic pathway and the extrinsic pathway, the cleavage of Bid to tBid was investigated, and there was no obvious tBid, indicating that there was no signal transfer from the extrinsic pathway to the intrinsic pathway. In another experiment, PTK7 expression in HCT 116 was initially suppressed using PTK7 siRNA, resulting in apoptosis in these cells. The ability of cell lysates to cleave the peptide substrates (Ac-AEVD-AFC) was tested as an indicator of caspase-10 activity. The results in show significant increase in caspase 10 activity in cells treated with PTK7 siRNA.
The activation of caspase-10 in apoptosis induced by knocking down of PTK7.
Protein p53 involvement in PTK7-knockdown-induced apoptosis
Protein p53 has been proved as a tumor suppressor protein in humans
, and HCT 116 cells express wide-type p53.
In order to study the involvement of p53 in the apoptosis induced by PTK7 knockdown, p53-null HCT 116 was used as the second cell line to carry out PTK7 knockdown and other related experiments. First, the PTK7 expression level without/with siRNA treatment was monitored by flow cytometry (). The p53-null HCT 116 cells express a high amount of PTK7 on the cell membrane, but after 48 hours of PTK7 siRNA transfection, the peak for anti-PTK7-PE in p53-null HCT 116 shifted back to the peak of the background control protein, anti-IgG-PE. This indicated that the PTK7 expression level in p53-null HCT 116 cells transfected with PTK7 siRNA was greatly decreased. Next, the number of live p53-null HCT 116 cells transfected with PTK7 siRNA was shown to be significantly different from that of untreated groups on day 4, demonstrating a significant inhibition of cell viability in p53-null HCT 116 cells by treating with PTK7 siRNA (). And in a BrdU incorporation experiment, silencing of PTK7 significantly inhibited BrdU incorporation in p53-null HCT 116 cells, suggesting a direct effect of PTK7 protein on cell proliferation (). On the other hand, the Annexin V/PI staining experiment showed that the PTK7 siRNA-treated p53-null HCT 116 showed significant increase in apoptotic and dead cells on day 4 compared with untreated, vehicle, and nonspecific siRNA control groups.
PTK7 expression and cell apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.
The apoptosis induced by PTK7 knockdown has been proved to be caspase-10 dependent in wild type HCT 116. So the apoptosis pathway in p53-null HCT 116 induced by PTK7 knockdown was further investigated. JC-1 experiment was monitored by both fluorescence microscopy () and flow cytometry (). Clearly, mitochondrial membrane potential decreased in p53-null HCT 116 cells treated with PTK7 siRNA. At the same time, a caspase inhibitor experiment was carried out using p53-null HCT 116 cells. As shown in , pancaspase inhibitor or caspase-10 inhibitor treatment inhibited the apoptosis induced by PTK7 knockdown compared to all other inhibitors, which indicated the apoptosis in p53-null HCT 116 cells induced by PTK7 knockdown was caspased-10 dependent.
Mitochondria and caspase-10 involvement in the apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.