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Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells.

Khumalo T, Ferreira E, Jovanovic K, Veale RB, Weiss SF - PLoS ONE (2015)

Bottom Line: This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay.Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1.These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, The Republic of South Africa (RSA).

ABSTRACT
Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

No MeSH data available.


Related in: MedlinePlus

LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.
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pone.0139584.g002: LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.

Mentions: High expression of LRP/LR tends to augment the aggressive behaviour of cancer cells by mediating metastasis, tumor angiogenesis and the ability to evade cell death [29, 30, 33, 34, 36]. The MDA-MB 231 breast and WHCO1 oesophageal cancer cells lines were no exception as Khumalo et al. illustrated enhanced metastatic ability accompanied by high expression levels of LRP/LR[29]. The results of this study paved way for the investigation of how the knockdown of the expression of LRP/LR would impact on the cellular viability of these cell lines as well as the non-metastatic MCF-7 breast cancer cell line. We used siRNA LAMR1 as well as esiRNA RPSA (MDA-MB 231 cells) to downregulate LRP by targeting the messenger RNA of LRP (siRNA-scr and esiRNA RLUC acted as negative controls). Densitometry analysis of the western blots revealed that no decrease in LRP was observed 24 h or 48 h after transfection of MDA-MB 231 cells with siRNA LAMR1 (Fig 1). However there was a significant decrease in LRP levels 72 h after transfection of WHCO1, MDA-MB 231 and MCF-7 cells (Fig 2). A 78%, 34% and 100% decrease in LRP expression was observed for the respective cell lines when compared to non-transfected negative controls set to a 100%. Western blot analysis of MDA-MB-231 cells transfected with esiRNA-RPSA showed a 26% downregulation of LRP (Fig 2).


Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells.

Khumalo T, Ferreira E, Jovanovic K, Veale RB, Weiss SF - PLoS ONE (2015)

LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4591328&req=5

pone.0139584.g002: LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.
Mentions: High expression of LRP/LR tends to augment the aggressive behaviour of cancer cells by mediating metastasis, tumor angiogenesis and the ability to evade cell death [29, 30, 33, 34, 36]. The MDA-MB 231 breast and WHCO1 oesophageal cancer cells lines were no exception as Khumalo et al. illustrated enhanced metastatic ability accompanied by high expression levels of LRP/LR[29]. The results of this study paved way for the investigation of how the knockdown of the expression of LRP/LR would impact on the cellular viability of these cell lines as well as the non-metastatic MCF-7 breast cancer cell line. We used siRNA LAMR1 as well as esiRNA RPSA (MDA-MB 231 cells) to downregulate LRP by targeting the messenger RNA of LRP (siRNA-scr and esiRNA RLUC acted as negative controls). Densitometry analysis of the western blots revealed that no decrease in LRP was observed 24 h or 48 h after transfection of MDA-MB 231 cells with siRNA LAMR1 (Fig 1). However there was a significant decrease in LRP levels 72 h after transfection of WHCO1, MDA-MB 231 and MCF-7 cells (Fig 2). A 78%, 34% and 100% decrease in LRP expression was observed for the respective cell lines when compared to non-transfected negative controls set to a 100%. Western blot analysis of MDA-MB-231 cells transfected with esiRNA-RPSA showed a 26% downregulation of LRP (Fig 2).

Bottom Line: This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay.Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1.These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, The Republic of South Africa (RSA).

ABSTRACT
Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

No MeSH data available.


Related in: MedlinePlus