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Down Regulation of CLDND1 Induces Apoptosis in Breast Cancer Cells.

Achari C, Winslow S, Larsson C - PLoS ONE (2015)

Bottom Line: Genes that were down regulated in all cell lines were further studied for survival-supporting effects.However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation.There is today little information on the function of CLDND1.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden.

ABSTRACT
Identification of targets for apoptosis induction is important to provide novel therapeutic approaches in breast cancer. Our earlier studies showed that down regulation of protein kinase C δ (PKCδ) induces death in breast cancer cells. In this study we set out to identify previously unrecognized apoptosis regulators in breast cancer cells. To identify candidates, global expression analysis with microarray was performed after down regulation of PKCδ in the basal-like breast cancer cell lines MDA-MB-231, MDA-MB-468 and BT-549. Genes that were down regulated in all cell lines were further studied for survival-supporting effects. The claudin-like CLDND1 was singled out since several independent siRNAs targeting CLDND1 induced cell death in several cell lines. The cell death induced by CLDND1 knockdown was caspase-dependent, suggesting induction of apoptosis. Nuclear fragmentation, cleavage of caspase-3 and PARP and release of cytochrome C from the mitochondria upon CLDND1 depletion demonstrated involvement of the intrinsic apoptotic pathway. Inhibition of MEK1/2 and JNK further potentiated the cell death induction by CLDND1 knockdown. However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation. A concomitant inhibition of MEK1/2 suppresses the ERK1/2 phosphorylation and markedly potentiates the cell death following CLDND1 siRNA treatment. There is today little information on the function of CLDND1. These data provide novel information on CLDND1 and highlight it as a novel survival factor in basal-like breast cancer cell lines.

No MeSH data available.


Related in: MedlinePlus

CLDND1 depletion decreases viability of different breast cancer cell lines.(A) mRNA expression of CLDND1 in various breast cancer cell lines. MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells were incubated under normal conditions for 24 h. Cells were subsequently harvested and were analyzed for mRNA expression of CLDND1 with real-time quantitative PCR. In B-E, MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells respectively, were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h. Cells were thereafter subjected to WST-1 assay. Data (mean ± S.E.M., n = 3) represent the amount of viable cells expressed as percent viable cells obtained under control conditions (B-E). Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01) compared with control cells.
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pone.0130300.g002: CLDND1 depletion decreases viability of different breast cancer cell lines.(A) mRNA expression of CLDND1 in various breast cancer cell lines. MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells were incubated under normal conditions for 24 h. Cells were subsequently harvested and were analyzed for mRNA expression of CLDND1 with real-time quantitative PCR. In B-E, MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells respectively, were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h. Cells were thereafter subjected to WST-1 assay. Data (mean ± S.E.M., n = 3) represent the amount of viable cells expressed as percent viable cells obtained under control conditions (B-E). Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01) compared with control cells.

Mentions: The role of CLDND1 in breast cancer cells was validated by transfecting MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells with three different siRNA oligonucleotides targeting CLDND1 (Fig 2B–2E). A marked reduction in cell viability, as measured by a WST-1 assay, was detected following treatment with all CLDND1 siRNAs in MDA-MB-231 cells (p<0.05 for siCLDND1-2 and p<0.01 for siCLDND1-1 and siCLDND1-3). For MDA-MB-468 and BT-549 cells less pronounced effects, but with the same tendency, could be seen (Fig 2B–2D). However none of three CLDND1 siRNAs had any substantial effect on MCF-7 cells (Fig 2E). We also measured the basal levels of CLDND1 mRNA in MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells by doing qPCR analysis of CLDND1 gene (Fig 2A). Expression of CLDND1 was much higher in MDA-MB-231 cells compared to MDA-MB-468, BT-549 and MCF-7 cells.


Down Regulation of CLDND1 Induces Apoptosis in Breast Cancer Cells.

Achari C, Winslow S, Larsson C - PLoS ONE (2015)

CLDND1 depletion decreases viability of different breast cancer cell lines.(A) mRNA expression of CLDND1 in various breast cancer cell lines. MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells were incubated under normal conditions for 24 h. Cells were subsequently harvested and were analyzed for mRNA expression of CLDND1 with real-time quantitative PCR. In B-E, MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells respectively, were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h. Cells were thereafter subjected to WST-1 assay. Data (mean ± S.E.M., n = 3) represent the amount of viable cells expressed as percent viable cells obtained under control conditions (B-E). Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01) compared with control cells.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470986&req=5

pone.0130300.g002: CLDND1 depletion decreases viability of different breast cancer cell lines.(A) mRNA expression of CLDND1 in various breast cancer cell lines. MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells were incubated under normal conditions for 24 h. Cells were subsequently harvested and were analyzed for mRNA expression of CLDND1 with real-time quantitative PCR. In B-E, MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells respectively, were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h. Cells were thereafter subjected to WST-1 assay. Data (mean ± S.E.M., n = 3) represent the amount of viable cells expressed as percent viable cells obtained under control conditions (B-E). Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01) compared with control cells.
Mentions: The role of CLDND1 in breast cancer cells was validated by transfecting MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells with three different siRNA oligonucleotides targeting CLDND1 (Fig 2B–2E). A marked reduction in cell viability, as measured by a WST-1 assay, was detected following treatment with all CLDND1 siRNAs in MDA-MB-231 cells (p<0.05 for siCLDND1-2 and p<0.01 for siCLDND1-1 and siCLDND1-3). For MDA-MB-468 and BT-549 cells less pronounced effects, but with the same tendency, could be seen (Fig 2B–2D). However none of three CLDND1 siRNAs had any substantial effect on MCF-7 cells (Fig 2E). We also measured the basal levels of CLDND1 mRNA in MDA-MB-231, MDA-MB-468, BT-549 and MCF-7 cells by doing qPCR analysis of CLDND1 gene (Fig 2A). Expression of CLDND1 was much higher in MDA-MB-231 cells compared to MDA-MB-468, BT-549 and MCF-7 cells.

Bottom Line: Genes that were down regulated in all cell lines were further studied for survival-supporting effects.However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation.There is today little information on the function of CLDND1.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden.

ABSTRACT
Identification of targets for apoptosis induction is important to provide novel therapeutic approaches in breast cancer. Our earlier studies showed that down regulation of protein kinase C δ (PKCδ) induces death in breast cancer cells. In this study we set out to identify previously unrecognized apoptosis regulators in breast cancer cells. To identify candidates, global expression analysis with microarray was performed after down regulation of PKCδ in the basal-like breast cancer cell lines MDA-MB-231, MDA-MB-468 and BT-549. Genes that were down regulated in all cell lines were further studied for survival-supporting effects. The claudin-like CLDND1 was singled out since several independent siRNAs targeting CLDND1 induced cell death in several cell lines. The cell death induced by CLDND1 knockdown was caspase-dependent, suggesting induction of apoptosis. Nuclear fragmentation, cleavage of caspase-3 and PARP and release of cytochrome C from the mitochondria upon CLDND1 depletion demonstrated involvement of the intrinsic apoptotic pathway. Inhibition of MEK1/2 and JNK further potentiated the cell death induction by CLDND1 knockdown. However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation. A concomitant inhibition of MEK1/2 suppresses the ERK1/2 phosphorylation and markedly potentiates the cell death following CLDND1 siRNA treatment. There is today little information on the function of CLDND1. These data provide novel information on CLDND1 and highlight it as a novel survival factor in basal-like breast cancer cell lines.

No MeSH data available.


Related in: MedlinePlus