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

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's t-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.

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. IMPLICATIONS: siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis.

IgG1-iS18 impedes the adhesive and invasive potential of early and late stage malignant melanoma cells.

The 37kDa/67kDa laminin receptor (LRP/LR) is a non-integrin laminin receptor which is overexpressed in tumorigenic cells and supports progression of cancer via promoting metastasis, angiogenesis and telomerase activity and impediment of apoptosis. The present study investigates the role of LRP/LR on the metastatic potential of early (A375) and late (A375SM) stage malignant melanoma cells. Flow cytometry revealed that both early and late stage malignant melanoma cells display high levels of LRP/LR on their cell surface. Flow cytometry and western blot analysis showed that late stage malignant melanoma cells display significantly higher total and cell surface LRP/LR levels in comparison to early stage malignant melanoma cells and the poorly invasive breast cancer (MCF-7) control cell line. Targeting LRP/LR using the LRP/LR specific antibody IgG1-iS18 resulted in a significant reduction of the adhesive potential to laminin-1 and the invasive potential through the 'ECM-simulating' Matrigel™ of both early and late stage malignant melanoma cells. Furthermore, Pearson's correlation coefficient confirmed that increased LRP levels correlate with the increased invasive and adhesive potential in early and late stage melanoma cells. Thus, blocking LRP/LR using the IgG1-iS18 antibody may therefore be a promising therapeutic strategy for early and late stage malignant melanoma treatment.

Anti-LRP/LR-specific antibody IgG1-iS18 impedes adhesion and invasion of pancreatic cancer and neuroblastoma cells.

BACKGROUND: Cancer has become a global burden due to its high incidence and mortality rates, with an estimated 14.1 million cancer cases reported worldwide in 2012 particularly as a result of metastasis. Metastasis involves two crucial steps: adhesion and invasion, and the non-integrin receptor; the 37-kDa/67-kDa laminin receptor precursor/ high affinity laminin receptor (LRP/LR) has been shown to be overexpressed on the surface of tumorigenic cells, thus being implicated in the enhancement of these two crucial steps. The current study investigated the role of LRP/LR on the aggressiveness of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells with respect to their adhesive and invasive potential. METHODS: AsPC-1 and IMR-32 cells were utilized as the experimental cell lines for the study. Cell surface LRP/LR levels were visualised and quantified on the experimental and control (MCF-7) cell lines via confocal microscopy and flow cytometry, respectively. Total LRP/LR levels in the cell lines were assessed by Western blotting and the adhesive and invasive potential of the above-mentioned cell lines was determined before and after supplementation with the anti-LRP/LR specific antibody IgG1-iS18. Statistical significance of the data was confirmed via the use of the two-tailed student's t-test and Pearson's correlation coefficient. RESULTS: Flow cytometry revealed that AsPC-1 and IMR-32 cells displayed significantly higher cell surface LRP/LR levels in comparison to the MCF-7 control cell line. However, Western blotting and subsequent densitometric analysis revealed that all three tumorigenic cell lines displayed no significant difference in total LRP/LR levels. The treatment of AsPC-1 and IMR-32 cells with IgG1-iS18 caused a significant reduction in the adhesive and invasive potential of the cells to laminin-1 and through the ECM-like Matrigel™, respectively. Pearson's correlation coefficients indicated a high correlation, thus suggesting a directly proportional relationship between cell surface LRP/LR levels and the adhesive and invasive potential of AsPC-1 and IMR-32 cells. CONCLUSION: These findings suggest that through the interference of the LRP/LR-laminin-1 interaction, the anti-LRP/LR specific antibody IgG1-iS18 may act as an alternative therapeutic tool for the treatment of metastatic pancreatic cancer and neuroblastoma.