Knock-down of LRP/LR promotes apoptosis in early and late stage colorectal carcinoma cells via caspase activation

Authenticated colorectal cancer cell lines SW-480 and DLD-1 were obtained from American Tissue Culture Collection (ATCC) with catalogue numbers ATCC® CCL-228 and ATCC® CCL-221, respectively. Both cancer cell lines were cultured in DMEM/Ham’s F-12 (1:1) (GE Lifesciences) together with 10% Fetal Calf Serum (FCS) (Capricorn Scientific) and 1% penicillin/streptomycin (Biowest). All cells remained at 37 °C with 5% CO₂ in a humidified incubator.

Cell counts were performed with the TC20™ cell counter (Biorad) and cells were seeded at a density depending on the experiment being performed. Cells were allowed to reach 50–70% confluency prior to transfection. Both cancer cell lines were transfected with ON-TARGETplus SMARTpool Human-RPSA (GE Dharmacon) (targeted towards LRP/LR) – this siRNA will be referred to as siRPSA #1 and esiRNA-RLUC (serving as the negative control) (Sigma). The appropriate amounts of DharmaFect transfection reagent (GE Dharmacon) and Mission transfection reagent (Sigma) were added to the cells, respectively. The cancer cell lines were likewise transfected with esiRNA-RPSA (Sigma) which is also targeted to LRP/LR (this siRNA will be referred to as siRPSA #2). Thereafter, cells were incubated for 72 h at 37 °C. This procedure was performed before any further experiments took place.

To determine siRNA-treated LRP/LR levels in the colorectal cancer cell lines, western blotting was performed. Cell lysates containing 10 μg of protein were separated on 12% sodium dodecyl sulphate polyacrylamide gels via electrophoresis (SDS-PAGE) (Bio-Rad). Thereafter, PVDF membranes (Pall Corporation) were soaked in methanol (Associate Chemical Enterprise, ACE) for 2 min followed by a 5-min incubation in transfer buffer. Proteins were transferred at 300 V via electro-blotting. The membranes were then blocked for an hour in 0.1% PBS-Tween in 3% BSA. Thereafter, the membranes were incubated with the IgG1-iS18 primary antibody which was diluted in blocking buffer (1:5000). The membranes were then washed three times in PBS-Tween (10 min for each wash) followed by an incubation in the appropriate secondary antibody diluted in blocking buffer (1:10000) for 1 h. The membrane was washed three more times with PBS-Tween before adding the chemiluminescent substrate (Biorad) to the membrane in order to detect proteins. In addition, 42 kDa β-actin served as a loading control. Finally, densitometric analysis was completed in order to quantify protein levels using ImageLab™ software.

The MTT assay is a valid assay for determining cell viability employed in various cancer studies [28‐30, 32‐35]. Before transfections took place, 1 × 10⁴ cells/ml SW-480 and DLD-1 cells were seeded on 24-well plates. After transfection, cells were incubated at 37 °C for 72 h, followed by the addition of 1 mg/ml of MTT [100 μg of MTT (Duchfei Biochemic) being dissolved in 1 X PBS (Gibco)] to all wells. This was followed by a further incubation at 37 °C for 2 h. Thereafter, the media from each well containing MTT was removed and 500 μl DMSO was added to dissolve the residual formazan crystals (Merck Millipore). The resultant absorbance was measured at 570 nm. This procedure was performed for controls as well which included: untreated cells, PCA (Protocatechuic acid) positive control (Aldrich Chemistry) treated cells as well as esiRNA-RLUC negative control treated cells.

In order to evaluate nuclear morphology post knock-down of LRP/LR via siRNA technology, confocal microscopy was used. Early and late stage colorectal cancer cells were seeded onto coverslips at a cell density of 1 × 10⁵ cells/ml. After transfection, the cells were fixed in 4% PFA (Associated Chemical Enterprise, ACE) for 15 min prior to 3 washes with PBS. Once remaining PBS was blotted off after washing, cells were then incubated with 0.1% Triton-X for 20 min for permeabilization of the cell membrane. The cells were then washed twice followed by the addition of DAPI nuclear stain (Sigma) diluted in PBS (1:100) onto each coverslip and incubated for 8 min in the dark. Once stained, the coverslips were washed twice in PBS prior to each coverslip being mounted on a microscope slide with fluoromount (Sigma). The microscope slides were left to set for 45 min in the dark, after which they were maintained at 4 °C. Note: untreated cells were used as a control, esiRNA-RLUC as a negative control and PCA as a positive control. Airyscan is a technique used to enhance confocal laser scanning microscopy. It has been shown that total resolution is improved by a factor of 1.7 in all spatial directions i.e. a resolution of 140 nm laterally and 400 nm axially can be achieved. The Airyscan was used to further analyse the nuclear morphological changes observed after LRP/LR down-regulation (Zeiss LSM 780).

This experiment was performed as per the manufacturer’s directions (Beckman Coulter). Both SW-480 and DLD-1 cells were seeded at a cell density of 2 × 10⁶ cells/ml before transfection. After 72 h incubation at 37 °C, cells were subjected to trypsinization with trypsin/EDTA (Biowest) followed by washes with cold PBS. Thereafter, cells were centrifuged at 5000 rpm for 5 min was performed, after which pellets were resuspended in 1X annexin-binding buffer (BD Sciences). Thereafter, 10 μl of Annexin V-FITC (BD Sciences) solution and 5 μl of PI viability dye were added to each cell suspension which was followed by a 15-min incubation on ice in the dark. Subsequently, 400 μl of ice-cold 1X annexin binding buffer was added to the samples for 30 min and all resulting cell suspensions were reviewed using the BD Accuri C6 flow cytometer. Note: esiRNA-RLUC was the negative control and PCA was the positive control.

Caspase-3,-8 and − 9 assays were completed as per the manufacturer’s directions (Merck Millipore). Cells were seeded at a cell density of 1 × 10⁶ cells/ml before transfection. Cells were then centrifuged at 1200 rpm for 10 min, followed by pellet resuspension in 50 μl of lysis buffer. The samples were then incubated for 10 min on ice, followed by a further centrifugation at 10000 x g for 5 min. While the pellet was discarded, the supernatant was placed into a new microcentrifuge tube and put on ice. Thereafter, a BCA™ assay was performed in order to obtain the supernatant’s protein concentration. This was followed by 200 μg of protein being diluted per sample, prior to being added to wells of a 96-well plate. Once this was completed, 20 μl of 5X assay buffer was added to every sample. Thereafter, 10 μl of peptide substrate was added followed by incubation at 37 °C for 2 h. Finally, the absorbance was read at 405 nm. Note: esiRNA-RLUC treated cells served as a negative control and PCA treated cells were used as a positive control.

To understand the effect LRP/LR expression has on early and late stage colorectal cancer cell viability, down-regulation of the receptor had to be performed. Once early (SW-480) and late (DLD-1) cells were transfected with siRPSA #1 (targeted towards the 37 kDa LRP mRNA), evaluation of Western blotting and densitometry was performed. Densitometry showed that LRP was significantly knocked down in both SW-480 and DLD-1 cells when transfected with siRPSA #1. The SW-480 and DLD-1 transfected cells exhibited a 75 and 78% decrease in LRP expression, respectively, when compared to cells that were not transfected, since these LRP levels were set to 100% (Fig. 1a and b). Additionally, to determine whether the reduction in LRP expression had resulted due to siRPSA #1-mediated LRP knock-down and not just an off-target effect, an alternative siRNA that targets another region of LRP was utilised. When comparing them to the non-transfected cells, both early (SW-480) and late (DLD-1) stage colorectal cancer cells displayed a knock-down of 72 and 61% in LRP expression, respectively (Fig. 1c and d). SW-480 and DLD-1 cells transfected with the esiRNA-RLUC showed no significant LRP knock-down when comparing them to non-transfected cells. Once LRP expression was successfully down-regulated using siRNA technology, its effect on the viability of both cell lines were observed. MTT assays were performed which showed that when SW-480 and DLD-1 cells were transfected with siRPSA #1, cell viability was significantly decreased in contrast to cells that were not transfected, indicating that siRNA-mediated knock-down of the receptor leads to reductions in cell viability in both early and late stage colorectal cancer cell lines. The MTT assays revealed that SW-480 and DLD-1 cells treated with siRPSA #1 displayed a 60 and 55% decrease, respectively, in comparison to the cells that were not transfected (Fig. 1g). Additional MTT assays were performed to evaluate whether siRPSA #2-mediated LRP knock-down also influenced the viability of SW-480 and DLD-1 cells. Post treatment of cells with siRPSA #2 was found to reduce the viability significantly by 44 and 89% in both SW-480 and DLD-1 cells, respectively, when comparing them to the cells which were not transfected (Fig. 1g). Additionally, treating SW-480 and DLD-1 cells with the negative control siRNA, esiRNA-RLUC, indicated no notable change in cell viability in comparison to cells that were not transfected (Fig. 1g).

To determine whether the decreases in cell viability after treatment with siRPSA #1 was caused by apoptotic induction, nuclear morphological changes were studied by confocal microscopy and Airyscan. siRPSA #1 treated early (SW-480) stage colorectal cancer cells exhibited nuclear morphological changes in the form of condensed nuclei and reduced nuclear size (Fig. 2d), when compared to the nuclei of cells that were not transfected (Fig. 2a). Late (DLD-1) stage colorectal cancer cells transfected with siRPSA #1 presented nuclear morphological changes such as cellular fragmentation into membrane-bound bodies, weakened membrane integrity and membrane blebbing and (Fig. 2h), in contrast to nuclei of cells that were not transfected (Fig. 2e). Membrane blebbing was confirmed for the DLD-1 cells by bright field microscopy (Additional file 1: Figure S1). Results obtained for siRPSA #1 treated cells were consistent with those of the positive control, PCA (Fig. 2c and g). In addition, upon treatment with esiRNA-RLUC, both cell lines did not reveal any morphological changes in nuclei, when comparing them to cells that were not transfected (Fig. 2b and f).

Due to confocal microscopy proposing that the knock-down of LRP expression in early (SW-480) and late (DLD-1) stage colorectal cancer cells leads to morphological changes of the nuclei (a key feature of cells undergoing apoptosis), Annexin-V/PI assays had to be performed to confirm this result quantitively.

SW-480 cells treated with siRPSA #1 revealed 36.6% of cells undergoing early apoptosis, while 44.1% of cells underwent late apoptosis (Fig. 3.1d), in contrast to cells that were not transfected (Fig. 3.1a). Transfected DLD-1 cells with siRPSA #1 resulted in 10.0% of cells undergoing early apoptosis while 74.3% of cells underwent late apoptosis (Fig. 3.1h) when compared to cells that were not transfected (Fig. 3.1e) Additionally, upon treatment with esiRNA-RLUC, SW-480 and DLD-1 cell lines did not undergo apoptosis (Fig. 3.1b and f). PCA positive control showed most cells underwent late apoptosis (Fig. 3.1c and g. Further statistical analysis confirmed a significant increase in apoptotic cells when treated with siRPSA #1, in contrast to non-transfected cells in both cell lines (Fig. 3.2).

For additional validation of apoptosis occurring in early (SW-480) and late (DLD-1) stage colorectal cancer cells once LRP has been down-regulated, caspase-3 activity assays were completed. Post transfection with siRPSA #1, SW-480 cells were found to have a significant 4-fold increase in caspase-3 activity, in comparison to cells that were not transfected (Fig. 4a). DLD-1 cells treated with siRPSA #1 displayed a 5-fold increase in caspase-3 activity in contrast to non-tranfected cells (Fig. 4a). Furthermore, no differences in caspase-3 activity was seen when both cell lines were treated with esiRNA-RLUC (Fig. 4a). PCA positive control displayed a 3-fold and 5-fold increase in caspase-3 activity was observed in SW-480 and DLD-1 cells, respectively (Fig. 4a).

Caspase-3 activation occurs through both apoptosis pathways (intrinsic and extrinsic) hence, further insight of how the receptor aids in tumourigenic cell survival was required; therefore caspase-8 and -9 activity assays were performed. These assays determine whether treatment with siRPSA #1 leads to the activation of the extrinsic pathway (facilitated through caspase-8) or the intrinsic pathway (facilitated through caspase-9) in each of the cell lines. SW-480 and DLD-1 cells transfected with siRPSA #1 were found to undergo a 4-fold increase in caspase-8 activity, in comparison to cells that were not transfected (Fig. 4b). Moreover, SW-480 cells and DLD-1 cells indicated a significant 7-fold and 4-fold increase in caspase-9 activity, respectively, in comparison to cells that were not transfected (Fig. 4c). Moreover, both cell lines transfected with esiRNA-RLUC displayed no differences in caspase-8 and -9 activity in comparison to cells that were not transfected (Fig. 4b).