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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

Silencing of CLDND1 induces death in various breast cancer cell lines.MDA-MB-231, MDA-MB-468, BT-549, MCF-7 cells were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h (A-H). Cells were then either subjected to Annexin V-APC staining and flow cytometry analysis (A-D) or analyzed for mRNA expression of CLDND1 with real-time quantitative PCR (E-H). Data in A-D (mean ± S.E.M., n = 3) represent percent AnnexinV-positive cells related to control conditions and in E-F (mean ± S.E.M., n = 2–3) represent CLDND1 mRNA levels normalized to control. Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01; ***, p<0.001) compared with control cells.
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pone.0130300.g003: Silencing of CLDND1 induces death in various breast cancer cell lines.MDA-MB-231, MDA-MB-468, BT-549, MCF-7 cells were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h (A-H). Cells were then either subjected to Annexin V-APC staining and flow cytometry analysis (A-D) or analyzed for mRNA expression of CLDND1 with real-time quantitative PCR (E-H). Data in A-D (mean ± S.E.M., n = 3) represent percent AnnexinV-positive cells related to control conditions and in E-F (mean ± S.E.M., n = 2–3) represent CLDND1 mRNA levels normalized to control. Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01; ***, p<0.001) compared with control cells.

Mentions: To validate that the decrease in viable cell number is due to increased cell death, an Annexin-V assay was done following treatment with the siRNAs. An increase in cell death as measured with Annexin V positivity was detected with CLDND1 depletion in MDA-MB-231 and MDA-MB-468 cells (Fig 3A and 3B). It was significant for two of the siRNAs (p<0.01 in MDA-MB-231 cells and p<0.05 in MDA-MB-468 cells) and a similar tendency was observed with the third one. For BT-549 cells a tendency to increased, albeit not significant, was seen with all siRNAs (Fig 3C). However, no increase in cell death was obtained in MCF-7 cells (Fig 3D). The knockdown of CLDND1 in each of these cells was confirmed with qPCR analysis of CLDND1 gene (Fig 3E–3H).


Down Regulation of CLDND1 Induces Apoptosis in Breast Cancer Cells.

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

Silencing of CLDND1 induces death in various breast cancer cell lines.MDA-MB-231, MDA-MB-468, BT-549, MCF-7 cells were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h (A-H). Cells were then either subjected to Annexin V-APC staining and flow cytometry analysis (A-D) or analyzed for mRNA expression of CLDND1 with real-time quantitative PCR (E-H). Data in A-D (mean ± S.E.M., n = 3) represent percent AnnexinV-positive cells related to control conditions and in E-F (mean ± S.E.M., n = 2–3) represent CLDND1 mRNA levels normalized to control. Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01; ***, p<0.001) compared with control cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130300.g003: Silencing of CLDND1 induces death in various breast cancer cell lines.MDA-MB-231, MDA-MB-468, BT-549, MCF-7 cells were transfected with three different siRNA oligonucleotides targeting CLDND1 or with a control siRNA for 72 h (A-H). Cells were then either subjected to Annexin V-APC staining and flow cytometry analysis (A-D) or analyzed for mRNA expression of CLDND1 with real-time quantitative PCR (E-H). Data in A-D (mean ± S.E.M., n = 3) represent percent AnnexinV-positive cells related to control conditions and in E-F (mean ± S.E.M., n = 2–3) represent CLDND1 mRNA levels normalized to control. Asterisks indicate statistically significant differences (*, p<0.05; **, p<0.01; ***, p<0.001) compared with control cells.
Mentions: To validate that the decrease in viable cell number is due to increased cell death, an Annexin-V assay was done following treatment with the siRNAs. An increase in cell death as measured with Annexin V positivity was detected with CLDND1 depletion in MDA-MB-231 and MDA-MB-468 cells (Fig 3A and 3B). It was significant for two of the siRNAs (p<0.01 in MDA-MB-231 cells and p<0.05 in MDA-MB-468 cells) and a similar tendency was observed with the third one. For BT-549 cells a tendency to increased, albeit not significant, was seen with all siRNAs (Fig 3C). However, no increase in cell death was obtained in MCF-7 cells (Fig 3D). The knockdown of CLDND1 in each of these cells was confirmed with qPCR analysis of CLDND1 gene (Fig 3E–3H).

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