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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

Changes in nuclear area after siRNA-mediated downregulation of LRP.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.
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pone.0139584.g007: Changes in nuclear area after siRNA-mediated downregulation of LRP.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.

Mentions: In addition to the observation of this fundamental hallmark of apoptosis, MCF-7, MDA-MB 231 and WHCO1 cells, transfected with siRNA-LAMR1 and stained with the fluorescent nuclear dye Hoechst 33324 exhibited nuclei constriction, loss of nuclear morphology and integrity when compared to siRNA-scr-transfected cells as indicated by the white arrows (Fig 6). A loss of nuclear morphology was exhibited by the crescent-shaped nuclei that are due to the collapse of chromatin as clearly observed in the 8mM PCA and siRNA-LAMR1 treated cell micrographs of MDA-MB 231 cells. siRNA-LAMR1 treated MCF-7 cells exhibited shrinked nuclei that form small balls that form apoptotic bodies whereas a majority of siRNA-LAMR1 treated WHCO1 cells underwent gradual chromatin condensation (Fig 6). The changes in nuclear morphology were quantified by measuring the nuclear area of the cells. Treatment of MCF-7, MDA-MB 231 and WHCO1 cells with 8 mm PCA as well as siRNA-LAMR1 furthermore caused a significant decrease in nuclear area as shown in Fig 7.


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)

Changes in nuclear area after siRNA-mediated downregulation of LRP.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139584.g007: Changes in nuclear area after siRNA-mediated downregulation of LRP.72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.
Mentions: In addition to the observation of this fundamental hallmark of apoptosis, MCF-7, MDA-MB 231 and WHCO1 cells, transfected with siRNA-LAMR1 and stained with the fluorescent nuclear dye Hoechst 33324 exhibited nuclei constriction, loss of nuclear morphology and integrity when compared to siRNA-scr-transfected cells as indicated by the white arrows (Fig 6). A loss of nuclear morphology was exhibited by the crescent-shaped nuclei that are due to the collapse of chromatin as clearly observed in the 8mM PCA and siRNA-LAMR1 treated cell micrographs of MDA-MB 231 cells. siRNA-LAMR1 treated MCF-7 cells exhibited shrinked nuclei that form small balls that form apoptotic bodies whereas a majority of siRNA-LAMR1 treated WHCO1 cells underwent gradual chromatin condensation (Fig 6). The changes in nuclear morphology were quantified by measuring the nuclear area of the cells. Treatment of MCF-7, MDA-MB 231 and WHCO1 cells with 8 mm PCA as well as siRNA-LAMR1 furthermore caused a significant decrease in nuclear area as shown in Fig 7.

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