Assessing the role of LRP/LR on the viability of pancreatic cancer and neuroblastoma cells through siRNA-mediated LRP/LR down-regulation

Abstract

Over the decades, cancer has become a global burden with alarmingly high incidence and mortality rates in both economically developed and developing countries. Characteristically, tumour cells exhibit an over-expression of the 37kDa/67kDa laminin receptor (LRP/LR) in comparison to their normal cell counterparts, with this receptor being implicated in several tumourigenic processes – importantly for the present study, the maintenance of cellular viability and the evasion of apoptosis. This present study aimed to elucidate the role of LRP/LR on the cellular viability of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. Flow cytometry revealed that both of these tumourigenic cell lines exhibited LRP/LR on their surface, with further analysis using median fluorescence intensity values showing that IMR-32 cells contain about 70% more cell-surface LRP/LR than AsPC-1 cells. Additionally, Western blotting and densitometry suggested that IMR-32 cells contained about 63% more total LRP/LR than AsPC-1 cells. Western blot analysis also revealed that targeting the mRNA of the 37kDa LRP using a LRP-specific siRNA (siRNA-LAMR1) in AsPC-1 and IMR-32 cells led to significant down-regulation of 90% and 71% in LRP expression, respectively. Consequently, MTT assays showed that LRP knockdown led to reductions of 82% and 65% in the viability of AsPC-1 and IMR-32 cells, respectively. Moreover, use of an alternative LRP-specific siRNA (esiRNA-RPSA) confirmed the specificity and excluded an off-target effect of siRNA-LAMR1 for LRP. BrdU assays revealed that knockdown of LRP reduced the proliferation of AsPC-1 and IMR-32 cells by 76% and 44%, respectively. Confocal microscopy indicated nuclear morphological changes suggestive of apoptosis as the form of cell death occuring in both cell lines after LRP down-regulation. This observation was confirmed using Annexin-V assays, which revealed that AsPC-1 cells underwent 44% more apoptosis than IMR-32 cells post LRP knockdown. Furthermore, caspase-3 activity assays revealed that both cell lines experienced apoptotic induction after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knockdown, IMR-32 cells undergo apoptosis solely via the extrinsic pathway, whilst AsPC-1 cells use both the intrinsic and extrinsic apoptotic pathways, possibly through a retaliatory feedback loop. Overall, LRP/LR is critical for the maintenance of the tumour cellular viability, making the receptor a promising therapeutic target and proposing the potential use of siRNA technology for treatment of pancreatic cancer and neuroblastoma.