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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

The effect of siRNA-mediated downregulation of LRP expression on cell membrane integrity.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Annexin-V FITC and 7-AAD then analysed by flow cytometry. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells revealed high FITC and 7-AAD signals indicative of cells undergoing early and late apoptosis, compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).
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pone.0139584.g005: The effect of siRNA-mediated downregulation of LRP expression on cell membrane integrity.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Annexin-V FITC and 7-AAD then analysed by flow cytometry. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells revealed high FITC and 7-AAD signals indicative of cells undergoing early and late apoptosis, compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).

Mentions: To elucidate a possible mechanism for the decrease in cellular viability upon knockdown of LRP in MCF-7, MDA-MB 231 and WHCO1 cancer cells, changes in the integrity of cell membrane and nuclear morphology were evaluated. Cells that were transfected with siRNA-LAMR1, siRNA-scr (negative control) and 8mM PCA (positive control) as previously described were labelled with Annexin V-FITC and 7-AAD. During apoptosis induction, cell integrity is lost and phospholipids such as phosphatidylserine (PS) become exposed at the cell surface. Annexin-V binds to PS with very high affinity thus labelling cells undergoing apoptosis when membrane blebbing occurs. 7-AAD distinguishes between early and late apoptotic cells. Indeed, the reduced cellular viability of breast MCF-7 and oesophageal WHCO1 cancer cells was due to the induction of apoptosis as depicted by the shift of cells from the bottom left quadrant (live cells) to the bottom right and top right quadrants (representing early and late apoptotic cells, respectively) (Fig 5). The MDA-MB 231 cells were observed to be undergoing late apoptosis as well as necrosis (Fig 5) after the knockdown of LRP expression.


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)

The effect of siRNA-mediated downregulation of LRP expression on cell membrane integrity.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Annexin-V FITC and 7-AAD then analysed by flow cytometry. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells revealed high FITC and 7-AAD signals indicative of cells undergoing early and late apoptosis, compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139584.g005: The effect of siRNA-mediated downregulation of LRP expression on cell membrane integrity.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Annexin-V FITC and 7-AAD then analysed by flow cytometry. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells revealed high FITC and 7-AAD signals indicative of cells undergoing early and late apoptosis, compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).
Mentions: To elucidate a possible mechanism for the decrease in cellular viability upon knockdown of LRP in MCF-7, MDA-MB 231 and WHCO1 cancer cells, changes in the integrity of cell membrane and nuclear morphology were evaluated. Cells that were transfected with siRNA-LAMR1, siRNA-scr (negative control) and 8mM PCA (positive control) as previously described were labelled with Annexin V-FITC and 7-AAD. During apoptosis induction, cell integrity is lost and phospholipids such as phosphatidylserine (PS) become exposed at the cell surface. Annexin-V binds to PS with very high affinity thus labelling cells undergoing apoptosis when membrane blebbing occurs. 7-AAD distinguishes between early and late apoptotic cells. Indeed, the reduced cellular viability of breast MCF-7 and oesophageal WHCO1 cancer cells was due to the induction of apoptosis as depicted by the shift of cells from the bottom left quadrant (live cells) to the bottom right and top right quadrants (representing early and late apoptotic cells, respectively) (Fig 5). The MDA-MB 231 cells were observed to be undergoing late apoptosis as well as necrosis (Fig 5) after the knockdown of LRP expression.

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