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

Knock-down of LRP/LR promotes apoptosis in early and late stage colorectal carcinoma cells via... Background: Cancer remains one of the leading causes of death around the world, where incidence and mortality rates are at a constant increase. Tumourigenic cells are characteristically seen to over-express the 37 kDa/67 kDa laminin receptor (LRP/LR) compared to their normal cell counterparts. This receptor has numerous roles in tumourigenesis including metastasis, angiogenic enhancement, telomerase activation, cell viability and apoptotic evasion. This study aimed to expose the role of LRP/LR on the cellular viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells. Methods: siRNA were used to down-regulate the expression of LRP/LR in SW-480 and DLD-1 cells which was assessed using western blotting. Subsequently, cell survival was evaluated using the MTT cell survival assay and confocal microscopy. Thereafter, Annexin V-FITC/PI staining and caspase activity assays were used to investigate the mechanism underlying the cell death observed upon LRP/LR knockdown. The data was analysed using Student’st- test with a confidence interval of 95%, with p-values of less than 0.05 seen as significant. Results: Here we reveal that siRNA-mediated knock-down of LRP led to notable decreases in cell viability through increased levels of apoptosis, apparent by compromised membrane integrity and significantly high caspase-3 activity. Down-regulated LRP resulted in a significant increase in caspase-8 and -9 activity in both cell lines. Conclusions: These findings show that the receptor is critically implicated in apoptosis and that LRP/LR down- regulation induces apoptosis in early and late stage colorectal cancer cells through both apoptotic pathways. Thus, this study suggests that siRNA-mediated knock-down of LRP could be a possible therapeutic strategy for the treatment of early and late stage colorectal carcinoma. Keywords: Colorectal cancer, Small interfering RNAs, Apoptosis, 37 kDa/67 kDa laminin receptor, LRP/LR, Therapeutics Background ranked as the 3rd most common cancer type with over Cancer remains one of the main causes of death around 1.4 million new cases in the year 2015 – contributing to the world, where incidence and mortality rates are at a 9.7% of the total number of cancer cases diagnosed, constant increase. According to the World Health including 774,000 cancer related deaths [1]. Due to the Organisation (WHO), over 14 million new cases were increasing prevalence and mortality rates of colorectal diagnosed in 2015, and 8.8 million cancer related deaths cancer, it is crucial to develop a novel treatment strategy were reported [1]. The current study focuses on a to combat this disease. particular cancer type known as colorectal cancer. In There are several intrinsic and extrinsic factors which South Africa, colorectal cancer is found to be the 5th contribute to normal cells transforming into cancerous most common cancer [2]. However, globally, it has been cells. Due to the complexity and diversity of neoplastic diseases, the collective term known as “the hallmarks of cancer” came about in order to provide a better under- * Correspondence: Stefan.weiss@wits.ac.za standing of this disease [3]. These hallmarks show that School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, Republic of South Africa © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vania et al. BMC Cancer (2018) 18:602 Page 2 of 11 tumour cells acquire several capabilities that their nor- apoptosis i.e. the intrinsic mitochondrial pathway and mal counterparts do not have, including: independent of the extrinsic death receptor pathway [31]. growth signals, resistance to anti-growth signals, unlim- Hence, the current study investigated whether ited replicative potential, tissue invasion and metastasis, siRNA-mediated knock-down of LRP/LR will reduce the continuous angiogenesis and apoptosis evasion [3]. In viability of early (SW-480) and late (DLD-1) stage colo- addition, recent studies have shown that cancerous rectal cancer cells. This study revealed that knock-down cells also require the help of a particular receptor of LRP/LR using siRNA technology significantly reduces known as the 37 kDa laminin receptor precursor/ the viability of early and late stage colorectal cancer 67 kDa laminin receptor (LRP/LR) to maintain their cells, and proposes that apoptosis is the cause for the tumourigenic state [4–9]. notable decreases in cellular viability. LRP/LR, also known as RPSA, is known to assist in numerous physiological processes [10, 11] . Moreover, Methods the receptor possesses a strong binding affinity for A detailed list of suppliers/manufacturers of antibodies, laminin-1, a ligand found in several non-collagenous gly- reagents and equipment used to carry out the following coproteins and is said to play critical roles in cell attach- experiments is given in the supplementary data section. ment, cell growth and differentiation [12], cell migration [13] and angiogenesis [14]. Hence, the interaction be- Cell culture and conditions tween LRP/LR and laminin-1 in is seen as an enhance- Authenticated colorectal cancer cell lines SW-480 and ment of tumour growth and progression [15]. In DLD-1 were obtained from American Tissue Culture addition, LRP/LR has also been seen to play several Collection (ATCC) with catalogue numbers ATCC® other roles such as maintaining ribosomal processing of CCL-228 and ATCC® CCL-221, respectively. Both cancer RNA [16], protein synthesis [17], cell cycle regulation cell lines were cultured in DMEM/Ham’s F-12 (1:1) (GE [17] and importantly, cell survival [18]. Lifesciences) together with 10% Fetal Calf Serum (FCS) Several studies have shown that LRP/LR contributes to (Capricorn Scientific) and 1% penicillin/streptomycin many other pathological conditions such as microbial in- (Biowest). All cells remained at 37 °C with 5% CO in a fections [19], neurological diseases including Alzheimer’s humidified incubator. disease [20–22], prion-related diseases [23], as well as numerous other cancer types [10]. Furthermore, Naidoo siRNA-mediated knock-down of the laminin receptor et al. has also shown that LRP/LR mediates telomerase (LRP/LR) activity by enhancing hTERT activity, thus, illustrating a Cell counts were performed with the TC20™ cell counter novel role for the receptor [24, 25]. Due to LRP/LR be- (Biorad) and cells were seeded at a density depending on ing involved in several of the aforementioned tumouri- the experiment being performed. Cells were allowed to genic processes, this prompted the investigation of the reach 50–70% confluency prior to transfection. Both receptor’s role in cellular viability and cell survival. One cancer cell lines were transfected with ON-TARGETplus study has revealed that through silencing LRP/LR via SMARTpool Human-RPSA (GE Dharmacon) (targeted siRNA technology, the viability of cervical (HeLa) [26], towards LRP/LR) – this siRNA will be referred to as liver (Hep3B) [27] and lung (A549) [26] cancer cells was siRPSA #1 and esiRNA-RLUC (serving as the negative reduced by means of apoptotic induction. Other studies control) (Sigma). The appropriate amounts of Dharma- indicated that the viability of breast (MCF-7 and Fect transfection reagent (GE Dharmacon) and Mission MDA-MB231) [28], oesophageal (WHC01) [28], neuro- transfection reagent (Sigma) were added to the cells, re- blastoma (IMR-32) [29], pancreatic (AsPC-1) [29] as well spectively. The cancer cell lines were likewise transfected as malignant melanoma cancer cells [30] was also re- with esiRNA-RPSA (Sigma) which is also targeted to duced through siRNA-mediated LRP/LR knockdown. LRP/LR (this siRNA will be referred to as siRPSA #2). Therefore, these studies show the vital role of LRP/LR in Thereafter, cells were incubated for 72 h at 37 °C. This apoptosis and maintaining tumour cell survival. procedure was performed before any further experi- Apoptosis is essential for several other processes ments took place. within organisms including: tissue homeostasis mainten- ance, normal development preservation, as well as dam- SDS-PAGE and western blotting aged cell elimination – all involving cells actively To determine siRNA-treated LRP/LR levels in the colo- committing suicide. Once cells undergo apoptosis, they rectal cancer cell lines, western blotting was performed. undergo several morphological and biochemical changes Cell lysates containing 10 μg of protein were separated on [31]. A biochemical change of importance to apoptosis 12% sodium dodecyl sulphate polyacrylamide gels via is the activation of caspases. These caspases may become electrophoresis (SDS-PAGE) (Bio-Rad). Thereafter, PVDF active through two key pathways and as a result induce membranes (Pall Corporation) were soaked in methanol Vania et al. BMC Cancer (2018) 18:602 Page 3 of 11 (Associate Chemical Enterprise, ACE) for 2 min followed dark, after which they were maintained at 4 °C. Note: by a 5-min incubation in transfer buffer. Proteins were untreated cells were used as a control, esiRNA-RLUC as a transferred at 300 V via electro-blotting. The membranes negative control and PCA as a positive control. Airyscan were then blocked for an hour in 0.1% PBS-Tween in 3% is a technique used to enhance confocal laser scanning BSA. Thereafter, the membranes were incubated with the microscopy. It has been shown that total resolution is IgG1-iS18 primary antibody which was diluted in blocking improved by a factor of 1.7 in all spatial directions i.e. a buffer (1:5000). The membranes were then washed three resolution of 140 nm laterally and 400 nm axially can be times in PBS-Tween (10 min for each wash) followed by achieved. The Airyscan was used to further analyse the an incubation in the appropriate secondary antibody di- nuclear morphological changes observed after LRP/LR luted in blocking buffer (1:10000) for 1 h. The membrane down-regulation (Zeiss LSM 780). was washed three more times with PBS-Tween before adding the chemiluminescent substrate (Biorad) to the Annexin V-FITC/7AAD assays membrane in order to detect proteins. In addition, 42 kDa This experiment was performed as per the manufac- β-actin served as a loading control. Finally, densitometric turer’s directions (Beckman Coulter). Both SW-480 and analysis was completed in order to quantify protein levels DLD-1 cells were seeded at a cell density of 2 × 10 using ImageLab™ software. cells/ml before transfection. After 72 h incubation at 37 °C, cells were subjected to trypsinization with tryp- MTT assay sin/EDTA (Biowest) followed by washes with cold PBS. The MTT assay is a valid assay for determining cell viabil- Thereafter, cells were centrifuged at 5000 rpm for 5 min ity employed in various cancer studies [28–30, 32–35]. was performed, after which pellets were resuspended in Before transfections took place, 1 × 10 cells/ml SW-480 1X annexin-binding buffer (BD Sciences). Thereafter, and DLD-1 cells were seeded on 24-well plates. After 10 μl of Annexin V-FITC (BD Sciences) solution and transfection, cells were incubated at 37 °C for 72 h, 5 μl of PI viability dye were added to each cell suspen- followed by the addition of 1 mg/ml of MTT [100 μgof sion which was followed by a 15-min incubation on ice MTT (Duchfei Biochemic) being dissolved in 1 X PBS in the dark. Subsequently, 400 μl of ice-cold 1X annexin (Gibco)] to all wells. This was followed by a further incu- binding buffer was added to the samples for 30 min and bation at 37 °C for 2 h. Thereafter, the media from each all resulting cell suspensions were reviewed using the well containing MTT was removed and 500 μlDMSO BD Accuri C6 flow cytometer. Note: esiRNA-RLUC was was added to dissolve the residual formazan crystals the negative control and PCA was the positive control. (Merck Millipore). The resultant absorbance was mea- sured at 570 nm. This procedure was performed for con- Caspase-3, − 8 and − 9 activation assays trols as well which included: untreated cells, PCA Caspase-3,-8 and − 9 assays were completed as per the (Protocatechuic acid) positive control (Aldrich Chemistry) manufacturer’s directions (Merck Millipore). Cells were treated cells as well as esiRNA-RLUC negative control seeded at a cell density of 1 × 10 cells/ml before trans- treated cells. fection. Cells were then centrifuged at 1200 rpm for 10 min, followed by pellet resuspension in 50 μl of lysis Assessment of nuclear morphological changes – Confocal buffer. The samples were then incubated for 10 min on microscopy with Airyscan ice, followed by a further centrifugation at 10000 x g for In order to evaluate nuclear morphology post 5 min. While the pellet was discarded, the supernatant knock-down of LRP/LR via siRNA technology, confocal was placed into a new microcentrifuge tube and put on microscopy was used. Early and late stage colorectal ice. Thereafter, a BCA™ assay was performed in order to cancer cells were seeded onto coverslips at a cell density obtain the supernatant’s protein concentration. This was of 1 × 10 cells/ml. After transfection, the cells were followed by 200 μg of protein being diluted per sample, fixed in 4% PFA (Associated Chemical Enterprise, ACE) prior to being added to wells of a 96-well plate. Once for 15 min prior to 3 washes with PBS. Once remaining this was completed, 20 μl of 5X assay buffer was added PBS was blotted off after washing, cells were then incu- to every sample. Thereafter, 10 μl of peptide substrate bated with 0.1% Triton-X for 20 min for permeabilization was added followed by incubation at 37 °C for 2 h. of the cell membrane. The cells were then washed twice Finally, the absorbance was read at 405 nm. Note: followed by the addition of DAPI nuclear stain (Sigma) di- esiRNA-RLUC treated cells served as a negative control luted in PBS (1:100) onto each coverslip and incubated for and PCA treated cells were used as a positive control. 8 min in the dark. Once stained, the coverslips were washed twice in PBS prior to each coverslip being Statistical evaluation mounted on a microscope slide with fluoromount (Sigma). In order for accurate data analysis, Student’s t-test The microscope slides were left to set for 45 min in the had to be utilized, with a confidence interval of 95%. Vania et al. BMC Cancer (2018) 18:602 Page 4 of 11 Furthermore, p-values greater than 0.05 were seen as by 44 and 89% in both SW-480 and DLD-1 cells, re- non-significant. To measure the degree of association spectively, when comparing them to the cells which between LRP/LR levels and apoptotic induction as were not transfected (Fig. 1g). Additionally, treating well as cellular viability, Pearson’s correlation coeffi- SW-480 and DLD-1 cells with the negative control cient was calculated. A positive coefficient shows a siRNA, esiRNA-RLUC, indicated no notable change in directly proportional relationship between the two cell viability in comparison to cells that were not variables (where values close to 1 indicates a highly transfected (Fig. 1g). positive correlation). Knock-down of LRP expression via siRNA technology Results leads to changes in nuclear morphology signifying siRNA technology successfully results in knock-down of apoptosis LRP expression and reductions in cellular viability in early To determine whether the decreases in cell viability after and late stage colorectal cancer cells treatment with siRPSA #1 was caused by apoptotic in- To understand the effect LRP/LR expression has on duction, nuclear morphological changes were studied by early and late stage colorectal cancer cell viability, confocal microscopy and Airyscan. siRPSA #1 treated down-regulation of the receptor had to be performed. early (SW-480) stage colorectal cancer cells exhibited Once early (SW-480) and late (DLD-1) cells were trans- nuclear morphological changes in the form of condensed fected with siRPSA #1 (targeted towards the 37 kDa LRP nuclei and reduced nuclear size (Fig. 2d), when com- mRNA), evaluation of Western blotting and densitom- pared to the nuclei of cells that were not transfected etry was performed. Densitometry showed that LRP was (Fig. 2a). Late (DLD-1) stage colorectal cancer cells significantly knocked down in both SW-480 and DLD-1 transfected with siRPSA #1 presented nuclear morpho- cells when transfected with siRPSA #1. The SW-480 and logical changes such as cellular fragmentation into DLD-1 transfected cells exhibited a 75 and 78% decrease membrane-bound bodies, weakened membrane integrity in LRP expression, respectively, when compared to cells and membrane blebbing and (Fig. 2h), in contrast to nu- that were not transfected, since these LRP levels were clei of cells that were not transfected (Fig. 2e). Mem- set to 100% (Fig. 1a and b). Additionally, to determine brane blebbing was confirmed for the DLD-1 cells by whether the reduction in LRP expression had resulted bright field microscopy (Additional file 1:Figure S1).Re- due to siRPSA #1-mediated LRP knock-down and not sults obtained for siRPSA #1 treated cells were consistent just an off-target effect, an alternative siRNA that targets with those of the positive control, PCA (Fig. 2c and g). In another region of LRP was utilised. When comparing addition, upon treatment with esiRNA-RLUC, both cell them to the non-transfected cells, both early lines did not reveal any morphological changes in nuclei, (SW-480) and late (DLD-1) stage colorectal cancer when comparing them to cells that were not transfected cells displayed a knock-down of 72 and 61% in LRP (Fig. 2b and f). expression, respectively (Fig. 1c and d). SW-480 and DLD-1 cells transfected with the esiRNA-RLUC siRNA-mediated knockdown of LRP expression induces showed no significant LRP knock-down when com- apoptosis in early and late stage colorectal cancer cells paring them to non-transfected cells. Once LRP ex- Due to confocal microscopy proposing that the pression was successfully down-regulated using siRNA knock-down of LRP expression in early (SW-480) and technology, its effect on the viability of both cell lines late (DLD-1) stage colorectal cancer cells leads to mor- were observed. MTT assays were performed which phological changes of the nuclei (a key feature of cells showed that when SW-480 and DLD-1 cells were undergoing apoptosis), Annexin-V/PI assays had to be transfected with siRPSA #1, cell viability was signifi- performed to confirm this result quantitively. cantly decreased in contrast to cells that were not SW-480 cells treated with siRPSA #1 revealed 36.6% of transfected, indicating that siRNA-mediated cells undergoing early apoptosis, while 44.1% of cells knock-down of the receptor leads to reductions in underwent late apoptosis (Fig. 3.1d), in contrast to cells cell viability in both early and late stage colorectal that were not transfected (Fig. 3.1a). Transfected DLD-1 cancer cell lines. The MTT assays revealed that cells with siRPSA #1 resulted in 10.0% of cells undergo- SW-480 and DLD-1 cells treated with siRPSA #1 dis- ing early apoptosis while 74.3% of cells underwent late played a 60 and 55% decrease, respectively, in comparison apoptosis (Fig. 3.1h) when compared to cells that were to the cells that were not transfected (Fig. 1g). Additional not transfected (Fig. 3.1e) Additionally, upon treatment MTT assays were performed to evaluate whether siRPSA with esiRNA-RLUC, SW-480 and DLD-1 cell lines did #2-mediated LRP knock-down also influenced the viability not undergo apoptosis (Fig. 3.1b and f). PCA positive of SW-480 and DLD-1 cells. Post treatment of cells with control showed most cells underwent late apoptosis siRPSA #2 was found to reduce the viability significantly (Fig. 3.1c and g. Further statistical analysis confirmed Vania et al. BMC Cancer (2018) 18:602 Page 5 of 11 Fig. 1 The effect of siRNA-mediated knock-down on LRP expression and the viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells. a), c) and e) Upon transfection of SW-480 with siRPSA #1 and siRPSA #2, significant 75 and 72% decreases in LRP expression levels was revealed, respectively, in contrast to cells that were not transfected. Densitometric analysis of LRP levels was performed where levels of the non-transfected cells were set to 100%. ***p = 0.0001, *p = 0.02 N.S.:p > 0.05. b), d) and f) siRPSA #1 and siRPSA #2 transfection in DLD-1 cells caused significant 79 and 61% decreases in LRP expression levels, respectively, when comparing them to cells that were not transfected. Densitometry displayed no significant difference in LRP expression between non-transfected and negative control esiRNA-RLUC transfected cells for both cell lines. ***p = 0.0005, *p = 0.02 N.S.:p > 0.05. g) MTT assays were performed to assess SW-480 and DLD-1 cell viability, upon treatment with siRPSA #1 and siRPSA #2. Non-transfected cell value were set to 100%. It was found that upon siRPSA #1 transfection, SW-480 and DLD-1 cells exhibited a significant decrease of 60 and 55% in cellular viability, respectively, in contrast to cells that were not transfected. It was also revealed that when the SW-480 and DLD-1 were transfected with siRPSA #2, there were significant reductions of 44 and 89% in cellular viability, respectively, when compared to non-transfected cells. Both cell lines showed no noteworthy differences in cell viability when treated with the negative control esiRNA-RLUC. PCA was used as the positive control. SW-480: siRPSA #1:***p = 0.0008, siRPSA #2:***p = 0.0004 DLD-1: siRPSA #1:*p = 0.01, siRPSA #2:***p = 0.0009, N.S. :p > 0.05, non-significant. All graphs represent an average of three biological and three technical repeats Vania et al. BMC Cancer (2018) 18:602 Page 6 of 11 Fig. 2 The effect of siRNA-mediated knock-down of LRP on nuclear morphology of early (SW-480) and late (DLD-1) stage colorectal cancer cells. Confocal microscopy with Airyscan analysis was completed to investigate changes in nuclear morphology upon siRNA treatment. a and e) Non-transfected SW-480 and DLD-1 cells displayed large nuclei with healthy membrane integrity. b and f) SW-480 and DLD-1 cells transfected with negative control esiRNA-RLUC showed parallel characteristics to the non-transfected cells with no changes in nuclear morphology. c) SW-480 cells treated with positive control PCA revealed nuclei that underwent apoptotic body formation. d) SW-480 cells transfected with siRPSA #1 exhibited nuclear shrinkage and condensed nuclei, proposing induction of apoptosis. g) DLD-1 cells treated with PCA displayed weakened membrane integrity and condensed nuclei. h) DLD-1 cells transfected with siRPSA #1 showed formation of apoptotic bodies and weakened membrane integrity. Images were obtained at a 630X magnification and Airy scan analysis was applied to each image. Scale bars are indicative of 20 μm a significant increase in apoptotic cells when treated intrinsic pathway (facilitated through caspase-9) in each with siRPSA #1, in contrast to non-transfected cells of the cell lines. SW-480 and DLD-1 cells transfected in both cell lines (Fig. 3.2). with siRPSA #1 were found to undergo a 4-fold increase in caspase-8 activity, in comparison to cells that were siRNA-mediated knock-down of LRP expression causes a not transfected (Fig. 4b). Moreover, SW-480 cells and notable increase in caspase-3 activity DLD-1 cells indicated a significant 7-fold and 4-fold in- For additional validation of apoptosis occurring in early crease in caspase-9 activity, respectively, in comparison (SW-480) and late (DLD-1) stage colorectal cancer cells to cells that were not transfected (Fig. 4c). Moreover, once LRP has been down-regulated, caspase-3 activity both cell lines transfected with esiRNA-RLUC displayed assays were completed. Post transfection with siRPSA no differences in caspase-8 and -9 activity in comparison #1, SW-480 cells were found to have a significant 4-fold to cells that were not transfected (Fig. 4b). increase in caspase-3 activity, in comparison to cells that were not transfected (Fig. 4a). DLD-1 cells treated with Discussion siRPSA #1 displayed a 5-fold increase in caspase-3 activ- LRP/LR has gained a large amount of interest due to the ity in contrast to non-tranfected cells (Fig. 4a). Further- many roles it plays in the cell. Particularly, the receptor’s more, no differences in caspase-3 activity was seen when over-expression in several cancer cell types as well as its both cell lines were treated with esiRNA-RLUC (Fig. 4a). contribution in tumourigenesis has become a target area PCA positive control displayed a 3-fold and 5-fold in- for research. LRP/LR has been seen to assist with several crease in caspase-3 activity was observed in SW-480 and tumourigenic processes including tumour adhesion and DLD-1 cells, respectively (Fig. 4a). invasion (metastasis), angiogenic enhancement as well as apoptotic evasion [3]. Additionally, since LRP/LR is not siRNA-mediated knock-down of LRP results in significant limited to the cell surface but also localized in the peri- increases in caspase-8 and caspase-9 activity in early and nuclear region, cytosol and nucleus, it is able to perform late stage colorectal cancer cells many intracellular and extracellular physiological roles Caspase-3 activation occurs through both apoptosis including maintaining cell viability, cell adhesion, cell pathways (intrinsic and extrinsic) hence, further insight growth and migration, cell cycle regulation, ribosomal of how the receptor aids in tumourigenic cell survival anchorage to microtubules, pre-rRNA processing and was required; therefore caspase-8 and -9 activity assays protein synthesis. Thus, cancerous cells are found to were performed. These assays determine whether treat- over-express LRP/LR, thereby exploiting these functions, ment with siRPSA #1 leads to the activation of the ex- resulting in the development of the abovementioned trinsic pathway (facilitated through caspase-8) or the tumourigenic processes. Moreover, a recent study Vania et al. BMC Cancer (2018) 18:602 Page 7 of 11 Fig. 3 3.1 Apoptotic induction in early (SW-480) and late (DLD-1) stage colorectal cancer cells post siRNA transfection. It was revealed that most of the non-transfected a) SW-480 and e) DLD-1 cells fall in the lower quadrant (Q1-LL) which is known to represent normal live cells. b) and f) SW-480 and DLD-1 cells treated with negative control esiRNA-RLUC mostly appeared in the lower quadrant indicating live cells. c) and g) Positive control PCA, revealed that most cells were found in the upper right quadrant (Q1-UR), representing SW-480 and DLD-1 cells undergoing late apoptosis, respectively. d) and h) Cells transfected with siRPSA #1 resulted in 36.6% of SW-480 cells (d) and 10% of DLD-1 cells (h) undergoing early apoptosis, which is depicted in the lower right quadrant (Q1-LR); while 44.1% of SW-480 cells and 74.3% of DLD-1 cells underwent late apoptosis. This indicates that upon treatment with siRPSA #1, a total of 80.7% of SW-480 cells and 84.3% DLD-1 cells underwent apoptosis. 3.2 Bar graph illustrating apoptotic induction in early (SW-480) and late (DLD-1) colorectal cancer cells after siRNA transfection. This graph displays an average of three experiments completed in triplicate. Percentages for each quadrant were pooled together and compared to one another for both cell lines. It was found that SW-480 and DLD-1 cells had a significant increase in early and late apoptosis when treated with siRPSA #1, in contrast to cells that were not transfected, and both cell lines were seen to undergo more late apoptosis than early apoptosis. SW-480: ***p = 8.92029E-06 (live), ***p = 0.0001 (early apoptosis), **p = 0.002; DLD-1: ***p = 1.97127E-05 (live), ***p = 3.72195E-05 (early apoptosis), ***p = 1.08522E-06 (late apoptosis) performed by Vania et al. showed that there were signifi- targeting the mRNA of the 37 kDa laminin receptor pre- cantly higher levels of the receptor in late (DLD-1) stage cursor (LRP) form, it was employed to down-regulate LRP colorectal cancer cells, compared to the early (SW-480) in this study (see Additional file 1: Table S1). Cells treated stage – indicating that LRP expression also increases in with siRPSA #1 resulted in significant decreases in LRP the course of malignant transformation [4]. down-regulation. Furthermore, a high correlation of 0.91 To gain insight into how LRP/LR maintains cell viability, for SW-480 and 0.96 for DLD-1 was observed between siRNA technology was used to knock-down LRP total levels of LRP before and after siRPSA #1 transfection expression (Fig. 1) and evaluate this effect on cell viability (Table 1). This high and positive correlation suggests that of SW-480 and DLD-1 cells. Due to siRPSA #1 only the level of LRP expression is indeed influenced by siRNA Vania et al. BMC Cancer (2018) 18:602 Page 8 of 11 Fig. 4 The effect of siRNA-mediated LRP knock-down on caspase-3, − 8 and − 9 activity in early (SW-480) and late (DLD-1) stage colorectal cancer cells. a) Upon treatment of SW-480 and DLD-1 cells with siRPSA #1, a significant 4-fold and 5-fold increase in caspase-3 activity was revealed, respectively, in comparison to cells that were not transfected (set to 100%). Both cell lines showed no significant difference in caspase-3 activity between cells transfected with negative control esiRNA-RLUC and non-transfected cells. PCA was used as a positive control. SW-480:***p = 0.0007 and DLD-1: **p = 0.0059. N.S: p > 0.05, non-significant. b) siRPSA #1 transfected SW-480 and DLD-1 cells both displayed a 4-fold significant increase in caspase-8 activity, compared to cells that were not transfected (set to 100%). Both cell lines showed no significant difference in caspase-8 activity between cells transfected with the esiRNA-RLUC and non-transfected cells. SW-480: **p = 0.0083 and DLD-1: **p = 0.002. N.S: p > 0.05, non-significant. C) SW-480 and DLD-1 cells transfected with siRPSA #1 showed a significant 7-fold increase and 4-fold increase, respectively, in contrast to cells that were not transfected. Both cell lines showed no significant difference in caspase-9 activity between cells transfected with the esiRNA-RLUC and non-transfected cells. SW-480:***p = 0.0001 and DLD-1: ***p = 0.0008. N.S.: p > 0.05, non-significant. This data represents three biological replicates which were completed in triplicate treatment i.e. lower levels of LRP expression prior to treat- To further investigate the receptor’s role in maintain- ment with siRNA leads to more LRP knockdown post ing cell viability, an MTT assay was employed to investi- treatment with siRNA. gate the effect of treatment with siRPSA #1 and siRPSA To validate that the observed knock-down was not #2 on cell viability. A significant decrease in viability was due to off target effects, SW-480 and DLD-1 cells were observed for both SW-480 and DLD-1 cells after LRP both treated with an alternative siRNA, siRPSA #2. This down-regulation (Fig. 1). These reductions in cellular siRNA targets a specific region of 37 kDa LRP mRNA viability correlate with the decreased levels of LRP ob- i.e. nucleotides 521–929 (Table 1). Upon treatment with served after siRNA-mediated down-regulation, signifying siRPSA #2, both cell lines showed significant decreases the receptor’s vital role in the survival of SW-480 and in LRP knock-down in contrast to cells that were not DLD-1 cells (Table 1). It has been discovered that LRP/ transfected (Fig. 1). These results validated that LRP was LR localised in the nucleus allows for chromosome sta- being down-regulated and was not just an off-target ef- bility maintenance via interactions with the Midkine fect. In addition, the correlation between total LRP levels heparin-binding growth factor; well-known for enhan- before and after siRPSA #2 treatment was found to be cing cell proliferation, migration and survival [36]. In high (sees Additional file 1: Table S2). addition, several cancer types are showed to have an Table 1 Assessment of correlation between levels of siRNA-mediated LRP knockdown and viability, apoptotic levels and caspase-3 activity of early (SW-480) and late (DLD-1) stage colorectal cancer cells, using Pearson’s correlation co-efficients (R) Cell line SW-480 DLD-1 Correlation between total LRP levels before and after siRPSA #1 transfection (R-value) 0.91 0.96 Correlation between siRPSA #1-mediated LRP knockdown and reduction in cell viability (R-value) 0.99 0.98 Correlation between siRPSA #2-mediated LRP knockdown and reduction in cell viability (R-value) 0.99 0.93 Correlation between total levels of apoptosis and total LRP levels after siRPSA #1 transfection (R-value) 0.99 0.98 Correlation between increases in caspase-3 activity and total LRP levels after siRPSA #1 (R-value) 0.94 0.93 Vania et al. BMC Cancer (2018) 18:602 Page 9 of 11 up-regulated expression of Midkine, which results in the is found to be significantly increased. Hence, to further promotion of cell survival factors as well as obstruction establish that apoptosis was indeed occurring and whether of apoptosis through caspase-3 inhibition [37]. However, caspases were activated after treatment with siRPSA #1, by targeting LRP expression through siRNA technology, caspase-3 assays were performed. Down-regulation of LRP/LR-Midkine interactions may decrease, and as a re- LRP caused a distinct increase in caspase-3 activity in sult decrease cell viability. SW-480 and DLD-1 cells when compared to the cells that To establish whether the observed decrease in were not transfected. Furthermore, the correlation be- SW-480 and DLD-1 cell viability post LRP knockdown tween the total levels of LRP after siRPSA #1-mediated was due to cell death caused by apoptosis, confocal mi- knockdown (Fig. 1) and increases in caspase-3 activity croscopy was used to evaluate nuclear morphology. Both (Fig. 4) was high in both cell lines (Table 1). These results cell lines revealed several changes in nuclear morph- noticeably point to the induction of apoptosis in SW-480 ology which were all characteristic of apoptosis includ- and DLD-1 cells due to the silencing of LRP though ing: nuclear condensation, reduced nuclear size and the siRNA technology. formation of membrane-bound bodies – when treated It has previously been shown that interactions between with siRPSA #1 (Fig. 2). It is known that nuclear struc- LRP/LR and focal adhesion kinase (FAK) are made pos- tures are maintained via the binding of histones to peri- sible via the binding of the receptor to laminin-1. Fur- nuclear and nuclear LRP/LR, thus when the receptor is thermore, LRP/LR-FAK interactions were seen to be down-regulated, loss of membrane integrity and dis- involved in activating cell signalling cascades such as torted nuclear morphology is evident [38]. MEK/ERK 1/2 and PI3-kinase/AKT as well as Although confocal microscopy provided a visual indi- up-regulating the anti-apoptotic protein, Bcl-2 [41]. This cation that apoptosis was occurring, additional quantifi- ultimately leads to the inhibition of apoptosis of cancer- cation and affirmation of apoptotic induction was ous cells. Hence, we suggest that silencing LRP/LR needed. This was made possible by means of Annexin through the use of siRNA technology as performed in V-FITC/PI assays. Non-transfected and negative control the current study, impedes the LRP/LR-FAK interaction, esiRNA-RLUC-transfected SW-480 and DLD-1 cells and in this way apoptosis is induced. Furthermore, LRP/ showed negative staining for Annexin-V – indicating live LR has been shown to have a direct relationship with cells. On the other hand, siRPSA #1-treated and the MAPK signalling pathway – where decreased levels PCA-treated SW-480 and DLD-1 cells exhibited positive of LRP causes a response in the pathway – resulting in staining for Annexin-V or Annexin-V and PI, indicating cell stress and ultimately, cell death [10]. Another reason early and late stage apoptosis, respectively. This shift of which could have led to apoptotic induction through Annexin-V staining from negative to positive shows that siRNA-mediated knock-down of LRP is the receptor’s siRPSA #1-mediated down-regulation in SW-480 and role in ribosomal processing. Research has shown that DLD-1 cells activates membrane asymmetry loss and a LRP/LR is involved in processing 21S pre-rRNA into membrane-flip reaction involving the externalization of mature 18S rRNA, also known as biogenesis of ribo- phosphatidylserine (PS) on the outer leaflet of the somes [16]. LRP/LR has also been shown to associate plasma membrane – allowing these cells to be taken up with pre-40S ribosomal subunits, providing nucleolar by phagocytes [39]. Thus, these findings together with exits for the subunits, thereby facilitating protein synthe- the nuclear morphological changes observed, confirm sis [42]. We propose that the LRP down-regulation that siRNA-mediated down-regulation of LRP/LR leads performed in this study may have hampered formation to the induction of apoptosis in SW-480 and DLD-1 of the ribosome as well as the resultant translation of cells. In addition, the correlation (Table 1) between total proteins required for correct cellular functioning, ultim- levels of LRP after siRPSA #1 treatment and total levels ately leading to apoptosis. Furthermore, LRP has also of apoptosis for both cell lines was found to be high been seen to play a key role in the cell cycle thus, (Figs. 1 and 3.1), further reiterating LRP expression af- down-regulating the receptor could have resulted in the fects cell viability and apoptosis. induction of G1-phase arrest in the colorectal cancer Sustantad et al. found that when LRP/LR is cells, aiding in apoptosis [10]. down-regulated via siRNA technology, not only did the Since both the MEK/ERK 1/2 and PI3-kinase/AKT cell cell viability of cervical cancer (HeLa) cells and lung signalling cascades are known to inhibit both apoptotic (A549) cancer cells decrease but caspase-3 activity was pathways, caspase-8 and caspase-9 assays were per- increased in both cell lines [27]. Caspase-3, an effector formed to determine if these caspases are activated upon caspase, is responsible in executing the hallmarks of siRPSA #1-mediated LRP/LR knock-down. Both early apoptosis which includes the afore-mentioned nuclear and late stage colorectal cancer cell lines were found to morphological changes by cleaving substrates [40]. have higher caspase-8 activity post transfection with Therefore, upon apoptotic induction, caspase-3 activity siRPSA #1, in contrast to cells that were not transfected. Vania et al. BMC Cancer (2018) 18:602 Page 10 of 11 Caspase-8 plays a vital role in the extrinsic apoptotic with uncompromised membrane integrity. C) and B) siRPSA #1-transfected signalling pathway through death receptors, thus it is and PCA (positive control) treated cells are found to have a reduced size together with compromised membrane integrity i.e. membrane blebbing suggested that siRNA-mediated LRP knock-down in- and condensed nuclei – all indicative of apoptosis occurring. Images were duces the apoptotic process in SW-480 and DLD-1 cells obtained at 200X magnification. Scale bars are indicative of 20 μm. extrinsically. Moreover, this study revealed that SW-480 Table S1. Sequence of Human-RPSA, esiRNA-RPSA and control siRNA-RLUC used for down-regulation of LRP/LR. Table S2. Pearson’scorrelation cells and DLD-1 also have increased in caspase-9 activity co-efficients (R) between total LRP levels prior to and post transfection after LRP down-regulation, in contrast to cells that were with esiRNA-RPSA (DOCX 425 kb) not transfected. Caspase-9 plays a critical role in the intrinsic apoptotic signalling pathway, proposing that Abbreviations siRNA-mediated LRP knock-down also initiates apop- ATCC: American type culture collection; BCA: Bicinchoninic acid; BSA: Bovine tosis in SW-480 and DLD-1 cells through the intrinsic serum albumin; CO : Carbon dioxide; DMEM: Dulbecco’s Modified Eagle’s medium; DMSO: Dimethyl sulfoxide; ERK: Extracellular signal-regulated ki- pathway. nases; FAK: Focal adhesion kinase; FCS: Fetal calf serum; FITC: Fluorescein The intrinsic and extrinsic pathways interconnect with isothiocyanate; HRP: Horseradish peroxidase; IgG: Immunoglobulin G; each other at several levels and can both be influenced kDa: Kilodaltons; LRP/LR: Laminin receptor precursor/ laminin receptor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; by similar factors. In fact, one study showed that PAGE: Polyacrylamide gel electrophoresis; PBS: Phosphate buffered saline; activated extrinsic caspase-8 stimulated the release of PCA: Protocatechuic acid; PI: Propidium iodide; PI3K: Phosphoinositide 3- cytochrome c and apoptosome formation and ultimately kinase; PS: Phosphatidyl serine; PVDF: Polyvinylidene fluoride; RLUC: Renilla luciferase; RNA: Ribonucleic acid; Rpm: Revolutions per minute; activation of the intrinsic pathway [43, 44]. A potential RPSA: Ribosomal protein SA; SDS: Sodium dodecyl sulfate; siRNA: Small reason as to why SW-480 and DLD-1 cells experience interfering RNA; TEMED: Tetramethylethylenediamine apoptosis through both apoptotic pathways may be that these colorectal cancer cells undergo a mechanism Acknowledgements known as retaliatory caspase activation where the two We thank Affimed Therapeutics GmbH, Heidelberg, Germany for providing antibody IgG1-iS18. We thank Carryn J. Chetty for guidance and knowledge apoptotic pathways are found to use a feedback amplifi- on the topic. cation loop in order to activate one another [45]. Specif- ically, activated caspase-9 initiates and proteolytically Funding cleaves caspase-3, also leading to caspase-8 activation This work is based upon research supported by the National Research [45, 46]. Moreover, due to SW-480 and DLD-1 cells Foundation (NRF), the Republic of South Africa (RSA). Grant Numbers 99061, 92745 and 109298. Any opinions, findings and conclusions or undergoing both apoptotic pathways, it can be said recommendations expressed in this material are those of the author(s), and that down-regulated LRP/LR possibly hampers both therefore, the National Research Foundation does not accept any liability in anti-apoptotic signalling pathways on account of the re- this regard thereto. Financial support was received from the South African Medical Research Council (SAMRC) under the Wits Common Epithelial duced interaction of phosphorylated FAK and LRP/LR. Cancer Research Centre (CECRC) grant. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s), and therefore, the SAMRC does not accept any liability in this Conclusions regard thereto. Financial support was further received from the Cancer This study shows that down-regulating LRP via siRNA Association of South Africa (CANSA). Any opinions, findings and conclusions technology significantly decreases the viability of early or recommendations expressed in this material are those of the author(s), and therefore, CANSA does not accept any liability in this regard thereto. (SW-480) and late (DLD-1) stage colorectal cancer cells through the induction of apoptosis. Moreover, SW-480 Availability of data and materials and DLD-1 cells underwent apoptosis through both All data generated or analysed during this study are included in this apoptotic pathways. It is possible that cell signalling cas- published article [and its Additional file 1]. cades are involved in inducing apoptosis, however, the exact mechanism is unclear. These findings demon- Authors’ contributions strates the critical function LRP/LR plays in maintaining SFTW conceptualised and designed the study. LV performed experiments. LV and TMR analysed the data. EF and SFTW edited the manuscript. All authors the viability of both early and late stage colorectal cancer have read and approved this version of the manuscript, and confirm that cells. In addition, these findings emphasize the thera- this is the case. peutic potential of siRNAs targeted against LRP, which could be used as a possible tool in treating early and late Ethics approval and consent to participate stage colorectal cancer. Not applicable. Competing interests Additional file The authors declare that they have no competing interests. Additional file 1: Figure S1. Late stage (DLD-1) colorectal cancer cells show membrane blebbing and reduced nuclei post transfection with Publisher’sNote siRPSA #1 using bright field microscopy. A) and B) Non-transfected and Springer Nature remains neutral with regard to jurisdictional claims in esiRNA-RLUC (negative control) transfected cells are found to be large published maps and institutional affiliations. Vania et al. BMC Cancer (2018) 18:602 Page 11 of 11 Received: 8 December 2017 Accepted: 18 May 2018 23. Leucht C, Simoneau S, Rey C, Vana K, Rieger R, Lasmézas CI, Weiss S. 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Knock-down of LRP/LR promotes apoptosis in early and late stage colorectal carcinoma cells via caspase activation

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Abstract

Background: Cancer remains one of the leading causes of death around the world, where incidence and mortality rates are at a constant increase. Tumourigenic cells are characteristically seen to over-express the 37 kDa/67 kDa laminin receptor (LRP/LR) compared to their normal cell counterparts. This receptor has numerous roles in tumourigenesis including metastasis, angiogenic enhancement, telomerase activation, cell viability and apoptotic evasion. This study aimed to expose the role of LRP/LR on the cellular viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells. Methods: siRNA were used to down-regulate the expression of LRP/LR in SW-480 and DLD-1 cells which was assessed using western blotting. Subsequently, cell survival was evaluated using the MTT cell survival assay and confocal microscopy. Thereafter, Annexin V-FITC/PI staining and caspase activity assays were used to investigate the mechanism underlying the cell death observed upon LRP/LR knockdown. The data was analysed using Student’st- test with a confidence interval of 95%, with p-values of less than 0.05 seen as significant. Results: Here we reveal that siRNA-mediated knock-down of LRP led to notable decreases in cell viability through increased levels of apoptosis, apparent by compromised membrane integrity and significantly high caspase-3 activity. Down-regulated LRP resulted in a significant increase in caspase-8 and -9 activity in both cell lines. Conclusions: These findings show that the receptor is critically implicated in apoptosis and that LRP/LR down- regulation induces apoptosis in early and late stage colorectal cancer cells through both apoptotic pathways. Thus, this study suggests that siRNA-mediated knock-down of LRP could be a possible therapeutic strategy for the treatment of early and late stage colorectal carcinoma. Keywords: Colorectal cancer, Small interfering RNAs, Apoptosis, 37 kDa/67 kDa laminin receptor, LRP/LR, Therapeutics Background ranked as the 3rd most common cancer type with over Cancer remains one of the main causes of death around 1.4 million new cases in the year 2015 – contributing to the world, where incidence and mortality rates are at a 9.7% of the total number of cancer cases diagnosed, constant increase. According to the World Health including 774,000 cancer related deaths [1]. Due to the Organisation (WHO), over 14 million new cases were increasing prevalence and mortality rates of colorectal diagnosed in 2015, and 8.8 million cancer related deaths cancer, it is crucial to develop a novel treatment strategy were reported [1]. The current study focuses on a to combat this disease. particular cancer type known as colorectal cancer. In There are several intrinsic and extrinsic factors which South Africa, colorectal cancer is found to be the 5th contribute to normal cells transforming into cancerous most common cancer [2]. However, globally, it has been cells. Due to the complexity and diversity of neoplastic diseases, the collective term known as “the hallmarks of cancer” came about in order to provide a better under- * Correspondence: Stefan.weiss@wits.ac.za standing of this disease [3]. These hallmarks show that School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, Republic of South Africa © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vania et al. BMC Cancer (2018) 18:602 Page 2 of 11 tumour cells acquire several capabilities that their nor- apoptosis i.e. the intrinsic mitochondrial pathway and mal counterparts do not have, including: independent of the extrinsic death receptor pathway [31]. growth signals, resistance to anti-growth signals, unlim- Hence, the current study investigated whether ited replicative potential, tissue invasion and metastasis, siRNA-mediated knock-down of LRP/LR will reduce the continuous angiogenesis and apoptosis evasion [3]. In viability of early (SW-480) and late (DLD-1) stage colo- addition, recent studies have shown that cancerous rectal cancer cells. This study revealed that knock-down cells also require the help of a particular receptor of LRP/LR using siRNA technology significantly reduces known as the 37 kDa laminin receptor precursor/ the viability of early and late stage colorectal cancer 67 kDa laminin receptor (LRP/LR) to maintain their cells, and proposes that apoptosis is the cause for the tumourigenic state [4–9]. notable decreases in cellular viability. LRP/LR, also known as RPSA, is known to assist in numerous physiological processes [10, 11] . Moreover, Methods the receptor possesses a strong binding affinity for A detailed list of suppliers/manufacturers of antibodies, laminin-1, a ligand found in several non-collagenous gly- reagents and equipment used to carry out the following coproteins and is said to play critical roles in cell attach- experiments is given in the supplementary data section. ment, cell growth and differentiation [12], cell migration [13] and angiogenesis [14]. Hence, the interaction be- Cell culture and conditions tween LRP/LR and laminin-1 in is seen as an enhance- Authenticated colorectal cancer cell lines SW-480 and ment of tumour growth and progression [15]. In DLD-1 were obtained from American Tissue Culture addition, LRP/LR has also been seen to play several Collection (ATCC) with catalogue numbers ATCC® other roles such as maintaining ribosomal processing of CCL-228 and ATCC® CCL-221, respectively. Both cancer RNA [16], protein synthesis [17], cell cycle regulation cell lines were cultured in DMEM/Ham’s F-12 (1:1) (GE [17] and importantly, cell survival [18]. Lifesciences) together with 10% Fetal Calf Serum (FCS) Several studies have shown that LRP/LR contributes to (Capricorn Scientific) and 1% penicillin/streptomycin many other pathological conditions such as microbial in- (Biowest). All cells remained at 37 °C with 5% CO in a fections [19], neurological diseases including Alzheimer’s humidified incubator. disease [20–22], prion-related diseases [23], as well as numerous other cancer types [10]. Furthermore, Naidoo siRNA-mediated knock-down of the laminin receptor et al. has also shown that LRP/LR mediates telomerase (LRP/LR) activity by enhancing hTERT activity, thus, illustrating a Cell counts were performed with the TC20™ cell counter novel role for the receptor [24, 25]. Due to LRP/LR be- (Biorad) and cells were seeded at a density depending on ing involved in several of the aforementioned tumouri- the experiment being performed. Cells were allowed to genic processes, this prompted the investigation of the reach 50–70% confluency prior to transfection. Both receptor’s role in cellular viability and cell survival. One cancer cell lines were transfected with ON-TARGETplus study has revealed that through silencing LRP/LR via SMARTpool Human-RPSA (GE Dharmacon) (targeted siRNA technology, the viability of cervical (HeLa) [26], towards LRP/LR) – this siRNA will be referred to as liver (Hep3B) [27] and lung (A549) [26] cancer cells was siRPSA #1 and esiRNA-RLUC (serving as the negative reduced by means of apoptotic induction. Other studies control) (Sigma). The appropriate amounts of Dharma- indicated that the viability of breast (MCF-7 and Fect transfection reagent (GE Dharmacon) and Mission MDA-MB231) [28], oesophageal (WHC01) [28], neuro- transfection reagent (Sigma) were added to the cells, re- blastoma (IMR-32) [29], pancreatic (AsPC-1) [29] as well spectively. The cancer cell lines were likewise transfected as malignant melanoma cancer cells [30] was also re- with esiRNA-RPSA (Sigma) which is also targeted to duced through siRNA-mediated LRP/LR knockdown. LRP/LR (this siRNA will be referred to as siRPSA #2). Therefore, these studies show the vital role of LRP/LR in Thereafter, cells were incubated for 72 h at 37 °C. This apoptosis and maintaining tumour cell survival. procedure was performed before any further experi- Apoptosis is essential for several other processes ments took place. within organisms including: tissue homeostasis mainten- ance, normal development preservation, as well as dam- SDS-PAGE and western blotting aged cell elimination – all involving cells actively To determine siRNA-treated LRP/LR levels in the colo- committing suicide. Once cells undergo apoptosis, they rectal cancer cell lines, western blotting was performed. undergo several morphological and biochemical changes Cell lysates containing 10 μg of protein were separated on [31]. A biochemical change of importance to apoptosis 12% sodium dodecyl sulphate polyacrylamide gels via is the activation of caspases. These caspases may become electrophoresis (SDS-PAGE) (Bio-Rad). Thereafter, PVDF active through two key pathways and as a result induce membranes (Pall Corporation) were soaked in methanol Vania et al. BMC Cancer (2018) 18:602 Page 3 of 11 (Associate Chemical Enterprise, ACE) for 2 min followed dark, after which they were maintained at 4 °C. Note: by a 5-min incubation in transfer buffer. Proteins were untreated cells were used as a control, esiRNA-RLUC as a transferred at 300 V via electro-blotting. The membranes negative control and PCA as a positive control. Airyscan were then blocked for an hour in 0.1% PBS-Tween in 3% is a technique used to enhance confocal laser scanning BSA. Thereafter, the membranes were incubated with the microscopy. It has been shown that total resolution is IgG1-iS18 primary antibody which was diluted in blocking improved by a factor of 1.7 in all spatial directions i.e. a buffer (1:5000). The membranes were then washed three resolution of 140 nm laterally and 400 nm axially can be times in PBS-Tween (10 min for each wash) followed by achieved. The Airyscan was used to further analyse the an incubation in the appropriate secondary antibody di- nuclear morphological changes observed after LRP/LR luted in blocking buffer (1:10000) for 1 h. The membrane down-regulation (Zeiss LSM 780). was washed three more times with PBS-Tween before adding the chemiluminescent substrate (Biorad) to the Annexin V-FITC/7AAD assays membrane in order to detect proteins. In addition, 42 kDa This experiment was performed as per the manufac- β-actin served as a loading control. Finally, densitometric turer’s directions (Beckman Coulter). Both SW-480 and analysis was completed in order to quantify protein levels DLD-1 cells were seeded at a cell density of 2 × 10 using ImageLab™ software. cells/ml before transfection. After 72 h incubation at 37 °C, cells were subjected to trypsinization with tryp- MTT assay sin/EDTA (Biowest) followed by washes with cold PBS. The MTT assay is a valid assay for determining cell viabil- Thereafter, cells were centrifuged at 5000 rpm for 5 min ity employed in various cancer studies [28–30, 32–35]. was performed, after which pellets were resuspended in Before transfections took place, 1 × 10 cells/ml SW-480 1X annexin-binding buffer (BD Sciences). Thereafter, and DLD-1 cells were seeded on 24-well plates. After 10 μl of Annexin V-FITC (BD Sciences) solution and transfection, cells were incubated at 37 °C for 72 h, 5 μl of PI viability dye were added to each cell suspen- followed by the addition of 1 mg/ml of MTT [100 μgof sion which was followed by a 15-min incubation on ice MTT (Duchfei Biochemic) being dissolved in 1 X PBS in the dark. Subsequently, 400 μl of ice-cold 1X annexin (Gibco)] to all wells. This was followed by a further incu- binding buffer was added to the samples for 30 min and bation at 37 °C for 2 h. Thereafter, the media from each all resulting cell suspensions were reviewed using the well containing MTT was removed and 500 μlDMSO BD Accuri C6 flow cytometer. Note: esiRNA-RLUC was was added to dissolve the residual formazan crystals the negative control and PCA was the positive control. (Merck Millipore). The resultant absorbance was mea- sured at 570 nm. This procedure was performed for con- Caspase-3, − 8 and − 9 activation assays trols as well which included: untreated cells, PCA Caspase-3,-8 and − 9 assays were completed as per the (Protocatechuic acid) positive control (Aldrich Chemistry) manufacturer’s directions (Merck Millipore). Cells were treated cells as well as esiRNA-RLUC negative control seeded at a cell density of 1 × 10 cells/ml before trans- treated cells. fection. Cells were then centrifuged at 1200 rpm for 10 min, followed by pellet resuspension in 50 μl of lysis Assessment of nuclear morphological changes – Confocal buffer. The samples were then incubated for 10 min on microscopy with Airyscan ice, followed by a further centrifugation at 10000 x g for In order to evaluate nuclear morphology post 5 min. While the pellet was discarded, the supernatant knock-down of LRP/LR via siRNA technology, confocal was placed into a new microcentrifuge tube and put on microscopy was used. Early and late stage colorectal ice. Thereafter, a BCA™ assay was performed in order to cancer cells were seeded onto coverslips at a cell density obtain the supernatant’s protein concentration. This was of 1 × 10 cells/ml. After transfection, the cells were followed by 200 μg of protein being diluted per sample, fixed in 4% PFA (Associated Chemical Enterprise, ACE) prior to being added to wells of a 96-well plate. Once for 15 min prior to 3 washes with PBS. Once remaining this was completed, 20 μl of 5X assay buffer was added PBS was blotted off after washing, cells were then incu- to every sample. Thereafter, 10 μl of peptide substrate bated with 0.1% Triton-X for 20 min for permeabilization was added followed by incubation at 37 °C for 2 h. of the cell membrane. The cells were then washed twice Finally, the absorbance was read at 405 nm. Note: followed by the addition of DAPI nuclear stain (Sigma) di- esiRNA-RLUC treated cells served as a negative control luted in PBS (1:100) onto each coverslip and incubated for and PCA treated cells were used as a positive control. 8 min in the dark. Once stained, the coverslips were washed twice in PBS prior to each coverslip being Statistical evaluation mounted on a microscope slide with fluoromount (Sigma). In order for accurate data analysis, Student’s t-test The microscope slides were left to set for 45 min in the had to be utilized, with a confidence interval of 95%. Vania et al. BMC Cancer (2018) 18:602 Page 4 of 11 Furthermore, p-values greater than 0.05 were seen as by 44 and 89% in both SW-480 and DLD-1 cells, re- non-significant. To measure the degree of association spectively, when comparing them to the cells which between LRP/LR levels and apoptotic induction as were not transfected (Fig. 1g). Additionally, treating well as cellular viability, Pearson’s correlation coeffi- SW-480 and DLD-1 cells with the negative control cient was calculated. A positive coefficient shows a siRNA, esiRNA-RLUC, indicated no notable change in directly proportional relationship between the two cell viability in comparison to cells that were not variables (where values close to 1 indicates a highly transfected (Fig. 1g). positive correlation). Knock-down of LRP expression via siRNA technology Results leads to changes in nuclear morphology signifying siRNA technology successfully results in knock-down of apoptosis LRP expression and reductions in cellular viability in early To determine whether the decreases in cell viability after and late stage colorectal cancer cells treatment with siRPSA #1 was caused by apoptotic in- To understand the effect LRP/LR expression has on duction, nuclear morphological changes were studied by early and late stage colorectal cancer cell viability, confocal microscopy and Airyscan. siRPSA #1 treated down-regulation of the receptor had to be performed. early (SW-480) stage colorectal cancer cells exhibited Once early (SW-480) and late (DLD-1) cells were trans- nuclear morphological changes in the form of condensed fected with siRPSA #1 (targeted towards the 37 kDa LRP nuclei and reduced nuclear size (Fig. 2d), when com- mRNA), evaluation of Western blotting and densitom- pared to the nuclei of cells that were not transfected etry was performed. Densitometry showed that LRP was (Fig. 2a). Late (DLD-1) stage colorectal cancer cells significantly knocked down in both SW-480 and DLD-1 transfected with siRPSA #1 presented nuclear morpho- cells when transfected with siRPSA #1. The SW-480 and logical changes such as cellular fragmentation into DLD-1 transfected cells exhibited a 75 and 78% decrease membrane-bound bodies, weakened membrane integrity in LRP expression, respectively, when compared to cells and membrane blebbing and (Fig. 2h), in contrast to nu- that were not transfected, since these LRP levels were clei of cells that were not transfected (Fig. 2e). Mem- set to 100% (Fig. 1a and b). Additionally, to determine brane blebbing was confirmed for the DLD-1 cells by whether the reduction in LRP expression had resulted bright field microscopy (Additional file 1:Figure S1).Re- due to siRPSA #1-mediated LRP knock-down and not sults obtained for siRPSA #1 treated cells were consistent just an off-target effect, an alternative siRNA that targets with those of the positive control, PCA (Fig. 2c and g). In another region of LRP was utilised. When comparing addition, upon treatment with esiRNA-RLUC, both cell them to the non-transfected cells, both early lines did not reveal any morphological changes in nuclei, (SW-480) and late (DLD-1) stage colorectal cancer when comparing them to cells that were not transfected cells displayed a knock-down of 72 and 61% in LRP (Fig. 2b and f). expression, respectively (Fig. 1c and d). SW-480 and DLD-1 cells transfected with the esiRNA-RLUC siRNA-mediated knockdown of LRP expression induces showed no significant LRP knock-down when com- apoptosis in early and late stage colorectal cancer cells paring them to non-transfected cells. Once LRP ex- Due to confocal microscopy proposing that the pression was successfully down-regulated using siRNA knock-down of LRP expression in early (SW-480) and technology, its effect on the viability of both cell lines late (DLD-1) stage colorectal cancer cells leads to mor- were observed. MTT assays were performed which phological changes of the nuclei (a key feature of cells showed that when SW-480 and DLD-1 cells were undergoing apoptosis), Annexin-V/PI assays had to be transfected with siRPSA #1, cell viability was signifi- performed to confirm this result quantitively. cantly decreased in contrast to cells that were not SW-480 cells treated with siRPSA #1 revealed 36.6% of transfected, indicating that siRNA-mediated cells undergoing early apoptosis, while 44.1% of cells knock-down of the receptor leads to reductions in underwent late apoptosis (Fig. 3.1d), in contrast to cells cell viability in both early and late stage colorectal that were not transfected (Fig. 3.1a). Transfected DLD-1 cancer cell lines. The MTT assays revealed that cells with siRPSA #1 resulted in 10.0% of cells undergo- SW-480 and DLD-1 cells treated with siRPSA #1 dis- ing early apoptosis while 74.3% of cells underwent late played a 60 and 55% decrease, respectively, in comparison apoptosis (Fig. 3.1h) when compared to cells that were to the cells that were not transfected (Fig. 1g). Additional not transfected (Fig. 3.1e) Additionally, upon treatment MTT assays were performed to evaluate whether siRPSA with esiRNA-RLUC, SW-480 and DLD-1 cell lines did #2-mediated LRP knock-down also influenced the viability not undergo apoptosis (Fig. 3.1b and f). PCA positive of SW-480 and DLD-1 cells. Post treatment of cells with control showed most cells underwent late apoptosis siRPSA #2 was found to reduce the viability significantly (Fig. 3.1c and g. Further statistical analysis confirmed Vania et al. BMC Cancer (2018) 18:602 Page 5 of 11 Fig. 1 The effect of siRNA-mediated knock-down on LRP expression and the viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells. a), c) and e) Upon transfection of SW-480 with siRPSA #1 and siRPSA #2, significant 75 and 72% decreases in LRP expression levels was revealed, respectively, in contrast to cells that were not transfected. Densitometric analysis of LRP levels was performed where levels of the non-transfected cells were set to 100%. ***p = 0.0001, *p = 0.02 N.S.:p > 0.05. b), d) and f) siRPSA #1 and siRPSA #2 transfection in DLD-1 cells caused significant 79 and 61% decreases in LRP expression levels, respectively, when comparing them to cells that were not transfected. Densitometry displayed no significant difference in LRP expression between non-transfected and negative control esiRNA-RLUC transfected cells for both cell lines. ***p = 0.0005, *p = 0.02 N.S.:p > 0.05. g) MTT assays were performed to assess SW-480 and DLD-1 cell viability, upon treatment with siRPSA #1 and siRPSA #2. Non-transfected cell value were set to 100%. It was found that upon siRPSA #1 transfection, SW-480 and DLD-1 cells exhibited a significant decrease of 60 and 55% in cellular viability, respectively, in contrast to cells that were not transfected. It was also revealed that when the SW-480 and DLD-1 were transfected with siRPSA #2, there were significant reductions of 44 and 89% in cellular viability, respectively, when compared to non-transfected cells. Both cell lines showed no noteworthy differences in cell viability when treated with the negative control esiRNA-RLUC. PCA was used as the positive control. SW-480: siRPSA #1:***p = 0.0008, siRPSA #2:***p = 0.0004 DLD-1: siRPSA #1:*p = 0.01, siRPSA #2:***p = 0.0009, N.S. :p > 0.05, non-significant. All graphs represent an average of three biological and three technical repeats Vania et al. BMC Cancer (2018) 18:602 Page 6 of 11 Fig. 2 The effect of siRNA-mediated knock-down of LRP on nuclear morphology of early (SW-480) and late (DLD-1) stage colorectal cancer cells. Confocal microscopy with Airyscan analysis was completed to investigate changes in nuclear morphology upon siRNA treatment. a and e) Non-transfected SW-480 and DLD-1 cells displayed large nuclei with healthy membrane integrity. b and f) SW-480 and DLD-1 cells transfected with negative control esiRNA-RLUC showed parallel characteristics to the non-transfected cells with no changes in nuclear morphology. c) SW-480 cells treated with positive control PCA revealed nuclei that underwent apoptotic body formation. d) SW-480 cells transfected with siRPSA #1 exhibited nuclear shrinkage and condensed nuclei, proposing induction of apoptosis. g) DLD-1 cells treated with PCA displayed weakened membrane integrity and condensed nuclei. h) DLD-1 cells transfected with siRPSA #1 showed formation of apoptotic bodies and weakened membrane integrity. Images were obtained at a 630X magnification and Airy scan analysis was applied to each image. Scale bars are indicative of 20 μm a significant increase in apoptotic cells when treated intrinsic pathway (facilitated through caspase-9) in each with siRPSA #1, in contrast to non-transfected cells of the cell lines. SW-480 and DLD-1 cells transfected in both cell lines (Fig. 3.2). with siRPSA #1 were found to undergo a 4-fold increase in caspase-8 activity, in comparison to cells that were siRNA-mediated knock-down of LRP expression causes a not transfected (Fig. 4b). Moreover, SW-480 cells and notable increase in caspase-3 activity DLD-1 cells indicated a significant 7-fold and 4-fold in- For additional validation of apoptosis occurring in early crease in caspase-9 activity, respectively, in comparison (SW-480) and late (DLD-1) stage colorectal cancer cells to cells that were not transfected (Fig. 4c). Moreover, once LRP has been down-regulated, caspase-3 activity both cell lines transfected with esiRNA-RLUC displayed assays were completed. Post transfection with siRPSA no differences in caspase-8 and -9 activity in comparison #1, SW-480 cells were found to have a significant 4-fold to cells that were not transfected (Fig. 4b). increase in caspase-3 activity, in comparison to cells that were not transfected (Fig. 4a). DLD-1 cells treated with Discussion siRPSA #1 displayed a 5-fold increase in caspase-3 activ- LRP/LR has gained a large amount of interest due to the ity in contrast to non-tranfected cells (Fig. 4a). Further- many roles it plays in the cell. Particularly, the receptor’s more, no differences in caspase-3 activity was seen when over-expression in several cancer cell types as well as its both cell lines were treated with esiRNA-RLUC (Fig. 4a). contribution in tumourigenesis has become a target area PCA positive control displayed a 3-fold and 5-fold in- for research. LRP/LR has been seen to assist with several crease in caspase-3 activity was observed in SW-480 and tumourigenic processes including tumour adhesion and DLD-1 cells, respectively (Fig. 4a). invasion (metastasis), angiogenic enhancement as well as apoptotic evasion [3]. Additionally, since LRP/LR is not siRNA-mediated knock-down of LRP results in significant limited to the cell surface but also localized in the peri- increases in caspase-8 and caspase-9 activity in early and nuclear region, cytosol and nucleus, it is able to perform late stage colorectal cancer cells many intracellular and extracellular physiological roles Caspase-3 activation occurs through both apoptosis including maintaining cell viability, cell adhesion, cell pathways (intrinsic and extrinsic) hence, further insight growth and migration, cell cycle regulation, ribosomal of how the receptor aids in tumourigenic cell survival anchorage to microtubules, pre-rRNA processing and was required; therefore caspase-8 and -9 activity assays protein synthesis. Thus, cancerous cells are found to were performed. These assays determine whether treat- over-express LRP/LR, thereby exploiting these functions, ment with siRPSA #1 leads to the activation of the ex- resulting in the development of the abovementioned trinsic pathway (facilitated through caspase-8) or the tumourigenic processes. Moreover, a recent study Vania et al. BMC Cancer (2018) 18:602 Page 7 of 11 Fig. 3 3.1 Apoptotic induction in early (SW-480) and late (DLD-1) stage colorectal cancer cells post siRNA transfection. It was revealed that most of the non-transfected a) SW-480 and e) DLD-1 cells fall in the lower quadrant (Q1-LL) which is known to represent normal live cells. b) and f) SW-480 and DLD-1 cells treated with negative control esiRNA-RLUC mostly appeared in the lower quadrant indicating live cells. c) and g) Positive control PCA, revealed that most cells were found in the upper right quadrant (Q1-UR), representing SW-480 and DLD-1 cells undergoing late apoptosis, respectively. d) and h) Cells transfected with siRPSA #1 resulted in 36.6% of SW-480 cells (d) and 10% of DLD-1 cells (h) undergoing early apoptosis, which is depicted in the lower right quadrant (Q1-LR); while 44.1% of SW-480 cells and 74.3% of DLD-1 cells underwent late apoptosis. This indicates that upon treatment with siRPSA #1, a total of 80.7% of SW-480 cells and 84.3% DLD-1 cells underwent apoptosis. 3.2 Bar graph illustrating apoptotic induction in early (SW-480) and late (DLD-1) colorectal cancer cells after siRNA transfection. This graph displays an average of three experiments completed in triplicate. Percentages for each quadrant were pooled together and compared to one another for both cell lines. It was found that SW-480 and DLD-1 cells had a significant increase in early and late apoptosis when treated with siRPSA #1, in contrast to cells that were not transfected, and both cell lines were seen to undergo more late apoptosis than early apoptosis. SW-480: ***p = 8.92029E-06 (live), ***p = 0.0001 (early apoptosis), **p = 0.002; DLD-1: ***p = 1.97127E-05 (live), ***p = 3.72195E-05 (early apoptosis), ***p = 1.08522E-06 (late apoptosis) performed by Vania et al. showed that there were signifi- targeting the mRNA of the 37 kDa laminin receptor pre- cantly higher levels of the receptor in late (DLD-1) stage cursor (LRP) form, it was employed to down-regulate LRP colorectal cancer cells, compared to the early (SW-480) in this study (see Additional file 1: Table S1). Cells treated stage – indicating that LRP expression also increases in with siRPSA #1 resulted in significant decreases in LRP the course of malignant transformation [4]. down-regulation. Furthermore, a high correlation of 0.91 To gain insight into how LRP/LR maintains cell viability, for SW-480 and 0.96 for DLD-1 was observed between siRNA technology was used to knock-down LRP total levels of LRP before and after siRPSA #1 transfection expression (Fig. 1) and evaluate this effect on cell viability (Table 1). This high and positive correlation suggests that of SW-480 and DLD-1 cells. Due to siRPSA #1 only the level of LRP expression is indeed influenced by siRNA Vania et al. BMC Cancer (2018) 18:602 Page 8 of 11 Fig. 4 The effect of siRNA-mediated LRP knock-down on caspase-3, − 8 and − 9 activity in early (SW-480) and late (DLD-1) stage colorectal cancer cells. a) Upon treatment of SW-480 and DLD-1 cells with siRPSA #1, a significant 4-fold and 5-fold increase in caspase-3 activity was revealed, respectively, in comparison to cells that were not transfected (set to 100%). Both cell lines showed no significant difference in caspase-3 activity between cells transfected with negative control esiRNA-RLUC and non-transfected cells. PCA was used as a positive control. SW-480:***p = 0.0007 and DLD-1: **p = 0.0059. N.S: p > 0.05, non-significant. b) siRPSA #1 transfected SW-480 and DLD-1 cells both displayed a 4-fold significant increase in caspase-8 activity, compared to cells that were not transfected (set to 100%). Both cell lines showed no significant difference in caspase-8 activity between cells transfected with the esiRNA-RLUC and non-transfected cells. SW-480: **p = 0.0083 and DLD-1: **p = 0.002. N.S: p > 0.05, non-significant. C) SW-480 and DLD-1 cells transfected with siRPSA #1 showed a significant 7-fold increase and 4-fold increase, respectively, in contrast to cells that were not transfected. Both cell lines showed no significant difference in caspase-9 activity between cells transfected with the esiRNA-RLUC and non-transfected cells. SW-480:***p = 0.0001 and DLD-1: ***p = 0.0008. N.S.: p > 0.05, non-significant. This data represents three biological replicates which were completed in triplicate treatment i.e. lower levels of LRP expression prior to treat- To further investigate the receptor’s role in maintain- ment with siRNA leads to more LRP knockdown post ing cell viability, an MTT assay was employed to investi- treatment with siRNA. gate the effect of treatment with siRPSA #1 and siRPSA To validate that the observed knock-down was not #2 on cell viability. A significant decrease in viability was due to off target effects, SW-480 and DLD-1 cells were observed for both SW-480 and DLD-1 cells after LRP both treated with an alternative siRNA, siRPSA #2. This down-regulation (Fig. 1). These reductions in cellular siRNA targets a specific region of 37 kDa LRP mRNA viability correlate with the decreased levels of LRP ob- i.e. nucleotides 521–929 (Table 1). Upon treatment with served after siRNA-mediated down-regulation, signifying siRPSA #2, both cell lines showed significant decreases the receptor’s vital role in the survival of SW-480 and in LRP knock-down in contrast to cells that were not DLD-1 cells (Table 1). It has been discovered that LRP/ transfected (Fig. 1). These results validated that LRP was LR localised in the nucleus allows for chromosome sta- being down-regulated and was not just an off-target ef- bility maintenance via interactions with the Midkine fect. In addition, the correlation between total LRP levels heparin-binding growth factor; well-known for enhan- before and after siRPSA #2 treatment was found to be cing cell proliferation, migration and survival [36]. In high (sees Additional file 1: Table S2). addition, several cancer types are showed to have an Table 1 Assessment of correlation between levels of siRNA-mediated LRP knockdown and viability, apoptotic levels and caspase-3 activity of early (SW-480) and late (DLD-1) stage colorectal cancer cells, using Pearson’s correlation co-efficients (R) Cell line SW-480 DLD-1 Correlation between total LRP levels before and after siRPSA #1 transfection (R-value) 0.91 0.96 Correlation between siRPSA #1-mediated LRP knockdown and reduction in cell viability (R-value) 0.99 0.98 Correlation between siRPSA #2-mediated LRP knockdown and reduction in cell viability (R-value) 0.99 0.93 Correlation between total levels of apoptosis and total LRP levels after siRPSA #1 transfection (R-value) 0.99 0.98 Correlation between increases in caspase-3 activity and total LRP levels after siRPSA #1 (R-value) 0.94 0.93 Vania et al. BMC Cancer (2018) 18:602 Page 9 of 11 up-regulated expression of Midkine, which results in the is found to be significantly increased. Hence, to further promotion of cell survival factors as well as obstruction establish that apoptosis was indeed occurring and whether of apoptosis through caspase-3 inhibition [37]. However, caspases were activated after treatment with siRPSA #1, by targeting LRP expression through siRNA technology, caspase-3 assays were performed. Down-regulation of LRP/LR-Midkine interactions may decrease, and as a re- LRP caused a distinct increase in caspase-3 activity in sult decrease cell viability. SW-480 and DLD-1 cells when compared to the cells that To establish whether the observed decrease in were not transfected. Furthermore, the correlation be- SW-480 and DLD-1 cell viability post LRP knockdown tween the total levels of LRP after siRPSA #1-mediated was due to cell death caused by apoptosis, confocal mi- knockdown (Fig. 1) and increases in caspase-3 activity croscopy was used to evaluate nuclear morphology. Both (Fig. 4) was high in both cell lines (Table 1). These results cell lines revealed several changes in nuclear morph- noticeably point to the induction of apoptosis in SW-480 ology which were all characteristic of apoptosis includ- and DLD-1 cells due to the silencing of LRP though ing: nuclear condensation, reduced nuclear size and the siRNA technology. formation of membrane-bound bodies – when treated It has previously been shown that interactions between with siRPSA #1 (Fig. 2). It is known that nuclear struc- LRP/LR and focal adhesion kinase (FAK) are made pos- tures are maintained via the binding of histones to peri- sible via the binding of the receptor to laminin-1. Fur- nuclear and nuclear LRP/LR, thus when the receptor is thermore, LRP/LR-FAK interactions were seen to be down-regulated, loss of membrane integrity and dis- involved in activating cell signalling cascades such as torted nuclear morphology is evident [38]. MEK/ERK 1/2 and PI3-kinase/AKT as well as Although confocal microscopy provided a visual indi- up-regulating the anti-apoptotic protein, Bcl-2 [41]. This cation that apoptosis was occurring, additional quantifi- ultimately leads to the inhibition of apoptosis of cancer- cation and affirmation of apoptotic induction was ous cells. Hence, we suggest that silencing LRP/LR needed. This was made possible by means of Annexin through the use of siRNA technology as performed in V-FITC/PI assays. Non-transfected and negative control the current study, impedes the LRP/LR-FAK interaction, esiRNA-RLUC-transfected SW-480 and DLD-1 cells and in this way apoptosis is induced. Furthermore, LRP/ showed negative staining for Annexin-V – indicating live LR has been shown to have a direct relationship with cells. On the other hand, siRPSA #1-treated and the MAPK signalling pathway – where decreased levels PCA-treated SW-480 and DLD-1 cells exhibited positive of LRP causes a response in the pathway – resulting in staining for Annexin-V or Annexin-V and PI, indicating cell stress and ultimately, cell death [10]. Another reason early and late stage apoptosis, respectively. This shift of which could have led to apoptotic induction through Annexin-V staining from negative to positive shows that siRNA-mediated knock-down of LRP is the receptor’s siRPSA #1-mediated down-regulation in SW-480 and role in ribosomal processing. Research has shown that DLD-1 cells activates membrane asymmetry loss and a LRP/LR is involved in processing 21S pre-rRNA into membrane-flip reaction involving the externalization of mature 18S rRNA, also known as biogenesis of ribo- phosphatidylserine (PS) on the outer leaflet of the somes [16]. LRP/LR has also been shown to associate plasma membrane – allowing these cells to be taken up with pre-40S ribosomal subunits, providing nucleolar by phagocytes [39]. Thus, these findings together with exits for the subunits, thereby facilitating protein synthe- the nuclear morphological changes observed, confirm sis [42]. We propose that the LRP down-regulation that siRNA-mediated down-regulation of LRP/LR leads performed in this study may have hampered formation to the induction of apoptosis in SW-480 and DLD-1 of the ribosome as well as the resultant translation of cells. In addition, the correlation (Table 1) between total proteins required for correct cellular functioning, ultim- levels of LRP after siRPSA #1 treatment and total levels ately leading to apoptosis. Furthermore, LRP has also of apoptosis for both cell lines was found to be high been seen to play a key role in the cell cycle thus, (Figs. 1 and 3.1), further reiterating LRP expression af- down-regulating the receptor could have resulted in the fects cell viability and apoptosis. induction of G1-phase arrest in the colorectal cancer Sustantad et al. found that when LRP/LR is cells, aiding in apoptosis [10]. down-regulated via siRNA technology, not only did the Since both the MEK/ERK 1/2 and PI3-kinase/AKT cell cell viability of cervical cancer (HeLa) cells and lung signalling cascades are known to inhibit both apoptotic (A549) cancer cells decrease but caspase-3 activity was pathways, caspase-8 and caspase-9 assays were per- increased in both cell lines [27]. Caspase-3, an effector formed to determine if these caspases are activated upon caspase, is responsible in executing the hallmarks of siRPSA #1-mediated LRP/LR knock-down. Both early apoptosis which includes the afore-mentioned nuclear and late stage colorectal cancer cell lines were found to morphological changes by cleaving substrates [40]. have higher caspase-8 activity post transfection with Therefore, upon apoptotic induction, caspase-3 activity siRPSA #1, in contrast to cells that were not transfected. Vania et al. BMC Cancer (2018) 18:602 Page 10 of 11 Caspase-8 plays a vital role in the extrinsic apoptotic with uncompromised membrane integrity. C) and B) siRPSA #1-transfected signalling pathway through death receptors, thus it is and PCA (positive control) treated cells are found to have a reduced size together with compromised membrane integrity i.e. membrane blebbing suggested that siRNA-mediated LRP knock-down in- and condensed nuclei – all indicative of apoptosis occurring. Images were duces the apoptotic process in SW-480 and DLD-1 cells obtained at 200X magnification. Scale bars are indicative of 20 μm. extrinsically. Moreover, this study revealed that SW-480 Table S1. Sequence of Human-RPSA, esiRNA-RPSA and control siRNA-RLUC used for down-regulation of LRP/LR. Table S2. Pearson’scorrelation cells and DLD-1 also have increased in caspase-9 activity co-efficients (R) between total LRP levels prior to and post transfection after LRP down-regulation, in contrast to cells that were with esiRNA-RPSA (DOCX 425 kb) not transfected. Caspase-9 plays a critical role in the intrinsic apoptotic signalling pathway, proposing that Abbreviations siRNA-mediated LRP knock-down also initiates apop- ATCC: American type culture collection; BCA: Bicinchoninic acid; BSA: Bovine tosis in SW-480 and DLD-1 cells through the intrinsic serum albumin; CO : Carbon dioxide; DMEM: Dulbecco’s Modified Eagle’s medium; DMSO: Dimethyl sulfoxide; ERK: Extracellular signal-regulated ki- pathway. nases; FAK: Focal adhesion kinase; FCS: Fetal calf serum; FITC: Fluorescein The intrinsic and extrinsic pathways interconnect with isothiocyanate; HRP: Horseradish peroxidase; IgG: Immunoglobulin G; each other at several levels and can both be influenced kDa: Kilodaltons; LRP/LR: Laminin receptor precursor/ laminin receptor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; by similar factors. In fact, one study showed that PAGE: Polyacrylamide gel electrophoresis; PBS: Phosphate buffered saline; activated extrinsic caspase-8 stimulated the release of PCA: Protocatechuic acid; PI: Propidium iodide; PI3K: Phosphoinositide 3- cytochrome c and apoptosome formation and ultimately kinase; PS: Phosphatidyl serine; PVDF: Polyvinylidene fluoride; RLUC: Renilla luciferase; RNA: Ribonucleic acid; Rpm: Revolutions per minute; activation of the intrinsic pathway [43, 44]. A potential RPSA: Ribosomal protein SA; SDS: Sodium dodecyl sulfate; siRNA: Small reason as to why SW-480 and DLD-1 cells experience interfering RNA; TEMED: Tetramethylethylenediamine apoptosis through both apoptotic pathways may be that these colorectal cancer cells undergo a mechanism Acknowledgements known as retaliatory caspase activation where the two We thank Affimed Therapeutics GmbH, Heidelberg, Germany for providing antibody IgG1-iS18. We thank Carryn J. Chetty for guidance and knowledge apoptotic pathways are found to use a feedback amplifi- on the topic. cation loop in order to activate one another [45]. Specif- ically, activated caspase-9 initiates and proteolytically Funding cleaves caspase-3, also leading to caspase-8 activation This work is based upon research supported by the National Research [45, 46]. Moreover, due to SW-480 and DLD-1 cells Foundation (NRF), the Republic of South Africa (RSA). Grant Numbers 99061, 92745 and 109298. Any opinions, findings and conclusions or undergoing both apoptotic pathways, it can be said recommendations expressed in this material are those of the author(s), and that down-regulated LRP/LR possibly hampers both therefore, the National Research Foundation does not accept any liability in anti-apoptotic signalling pathways on account of the re- this regard thereto. Financial support was received from the South African Medical Research Council (SAMRC) under the Wits Common Epithelial duced interaction of phosphorylated FAK and LRP/LR. Cancer Research Centre (CECRC) grant. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s), and therefore, the SAMRC does not accept any liability in this Conclusions regard thereto. Financial support was further received from the Cancer This study shows that down-regulating LRP via siRNA Association of South Africa (CANSA). Any opinions, findings and conclusions technology significantly decreases the viability of early or recommendations expressed in this material are those of the author(s), and therefore, CANSA does not accept any liability in this regard thereto. (SW-480) and late (DLD-1) stage colorectal cancer cells through the induction of apoptosis. Moreover, SW-480 Availability of data and materials and DLD-1 cells underwent apoptosis through both All data generated or analysed during this study are included in this apoptotic pathways. It is possible that cell signalling cas- published article [and its Additional file 1]. cades are involved in inducing apoptosis, however, the exact mechanism is unclear. These findings demon- Authors’ contributions strates the critical function LRP/LR plays in maintaining SFTW conceptualised and designed the study. LV performed experiments. LV and TMR analysed the data. EF and SFTW edited the manuscript. All authors the viability of both early and late stage colorectal cancer have read and approved this version of the manuscript, and confirm that cells. In addition, these findings emphasize the thera- this is the case. peutic potential of siRNAs targeted against LRP, which could be used as a possible tool in treating early and late Ethics approval and consent to participate stage colorectal cancer. Not applicable. Competing interests Additional file The authors declare that they have no competing interests. Additional file 1: Figure S1. Late stage (DLD-1) colorectal cancer cells show membrane blebbing and reduced nuclei post transfection with Publisher’sNote siRPSA #1 using bright field microscopy. A) and B) Non-transfected and Springer Nature remains neutral with regard to jurisdictional claims in esiRNA-RLUC (negative control) transfected cells are found to be large published maps and institutional affiliations. Vania et al. BMC Cancer (2018) 18:602 Page 11 of 11 Received: 8 December 2017 Accepted: 18 May 2018 23. Leucht C, Simoneau S, Rey C, Vana K, Rieger R, Lasmézas CI, Weiss S. 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BMC CancerSpringer Journals

Published: May 29, 2018

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