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Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways.

Siddiqa A, Long LM, Li L, Marciniak RA, Kazhdan I - BMC Cancer (2008)

Bottom Line: We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells.Additionally we show that survivin up-regulation is not at transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. siddiqa@uthscsa.edu

ABSTRACT

Background: The oncoprotein HER-2 is over-expressed and/or has undergone gene amplification in between 20 to 30% of breast and ovarian cancers. HER-2 amplified breast cancer is associated with a poor prognosis and increased resistance to chemo- and hormonal therapy. Data supporting the transforming potential of HER-2 are irrefutable but the mechanism by which HER-2 contributes to this process is complex and a unified model of HER2-induced increased cell proliferation and survival has not emerged.To understand the initial event(s) that take place by HER-2 over expression, we studied the effect of short term induction of HER-2 expression in the MCF7 breast cancer cell line.

Methods: We examined the modulation of apoptotic pathways by tetracycline-regulated HER-2 expression for 48 hrs in the MCF7 breast cancer cell line. Specific inhibitors were used to determine signalling pathways that are required for HER-2 induced up-regulation of survivin.

Results: Tetracycline regulated short term over expression of HER-2 in the MCF7 cell line increased the antiapoptotic proteins Bcl-2 and survivin levels. Significant increase of extracellular signal-related kinase (ERK) activation but not AKT1, AKT2 and STAT3 was observed in HER-2 over-expressing MCF7 cells. Specific inhibitors of ERK, and phosphoinositide-3 kinase (PI3K), inhibited the HER-2 induced up-regulation of survivin. We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.

Conclusion: Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells. We determined that survivin is up-regulated via ERK activation and PI3K signalling. Additionally we show that survivin up-regulation is not at transcriptional level. These data provide insight into the mechanism(s) by which induction of HER-2 over expression up-regulates survivin and Bcl-2 and identifies new targets for therapy of breast cancer.

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Related in: MedlinePlus

Activity of NF-kappaB in control and doxycycline-treated MCF7-HER2 cells. MCF7-HER2 cells were plated in duplicate wells at a concentration of 5 × 104 cells per well in 24 well plates. Doxycycline (Dox) or saline (control) was added 16 hours later and incubation continued for additional 48 hours, after which cells received fresh serum-containing medium with or without Dox. Cells were then co-transfected with NF-kappaB-RE-Luc plasmid and pRL-TK, which contains Renilla luciferase sequence under control of TK promoter. 48 hours later cells were harvested and dual-luciferase assay was performed per manufacturer's instructions. Data are presented as the ratio of Firefly/Renilla luciferase actvity × 100.
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Figure 6: Activity of NF-kappaB in control and doxycycline-treated MCF7-HER2 cells. MCF7-HER2 cells were plated in duplicate wells at a concentration of 5 × 104 cells per well in 24 well plates. Doxycycline (Dox) or saline (control) was added 16 hours later and incubation continued for additional 48 hours, after which cells received fresh serum-containing medium with or without Dox. Cells were then co-transfected with NF-kappaB-RE-Luc plasmid and pRL-TK, which contains Renilla luciferase sequence under control of TK promoter. 48 hours later cells were harvested and dual-luciferase assay was performed per manufacturer's instructions. Data are presented as the ratio of Firefly/Renilla luciferase actvity × 100.

Mentions: Stimulation of a wide variety of cell-surface receptors leads directly to NF-κB activation and a rapid change in gene expression. To determine whether NF-kappaB is activated in response to induction of HER-2 in MCF7 cells we transiently transfected control and doxycycline-treated MCF7-HER2 cells with a plasmid containing firefly luciferase sequence driven by five tandem consensus NF-kappaB elements. As demonstrated in Figure 6, there was no significant increase in NF-kappaB-mediated luciferase activity in cells with induced HER-2 overexpression as compared to their uninduced counterparts.


Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways.

Siddiqa A, Long LM, Li L, Marciniak RA, Kazhdan I - BMC Cancer (2008)

Activity of NF-kappaB in control and doxycycline-treated MCF7-HER2 cells. MCF7-HER2 cells were plated in duplicate wells at a concentration of 5 × 104 cells per well in 24 well plates. Doxycycline (Dox) or saline (control) was added 16 hours later and incubation continued for additional 48 hours, after which cells received fresh serum-containing medium with or without Dox. Cells were then co-transfected with NF-kappaB-RE-Luc plasmid and pRL-TK, which contains Renilla luciferase sequence under control of TK promoter. 48 hours later cells were harvested and dual-luciferase assay was performed per manufacturer's instructions. Data are presented as the ratio of Firefly/Renilla luciferase actvity × 100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2386479&req=5

Figure 6: Activity of NF-kappaB in control and doxycycline-treated MCF7-HER2 cells. MCF7-HER2 cells were plated in duplicate wells at a concentration of 5 × 104 cells per well in 24 well plates. Doxycycline (Dox) or saline (control) was added 16 hours later and incubation continued for additional 48 hours, after which cells received fresh serum-containing medium with or without Dox. Cells were then co-transfected with NF-kappaB-RE-Luc plasmid and pRL-TK, which contains Renilla luciferase sequence under control of TK promoter. 48 hours later cells were harvested and dual-luciferase assay was performed per manufacturer's instructions. Data are presented as the ratio of Firefly/Renilla luciferase actvity × 100.
Mentions: Stimulation of a wide variety of cell-surface receptors leads directly to NF-κB activation and a rapid change in gene expression. To determine whether NF-kappaB is activated in response to induction of HER-2 in MCF7 cells we transiently transfected control and doxycycline-treated MCF7-HER2 cells with a plasmid containing firefly luciferase sequence driven by five tandem consensus NF-kappaB elements. As demonstrated in Figure 6, there was no significant increase in NF-kappaB-mediated luciferase activity in cells with induced HER-2 overexpression as compared to their uninduced counterparts.

Bottom Line: We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells.Additionally we show that survivin up-regulation is not at transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. siddiqa@uthscsa.edu

ABSTRACT

Background: The oncoprotein HER-2 is over-expressed and/or has undergone gene amplification in between 20 to 30% of breast and ovarian cancers. HER-2 amplified breast cancer is associated with a poor prognosis and increased resistance to chemo- and hormonal therapy. Data supporting the transforming potential of HER-2 are irrefutable but the mechanism by which HER-2 contributes to this process is complex and a unified model of HER2-induced increased cell proliferation and survival has not emerged.To understand the initial event(s) that take place by HER-2 over expression, we studied the effect of short term induction of HER-2 expression in the MCF7 breast cancer cell line.

Methods: We examined the modulation of apoptotic pathways by tetracycline-regulated HER-2 expression for 48 hrs in the MCF7 breast cancer cell line. Specific inhibitors were used to determine signalling pathways that are required for HER-2 induced up-regulation of survivin.

Results: Tetracycline regulated short term over expression of HER-2 in the MCF7 cell line increased the antiapoptotic proteins Bcl-2 and survivin levels. Significant increase of extracellular signal-related kinase (ERK) activation but not AKT1, AKT2 and STAT3 was observed in HER-2 over-expressing MCF7 cells. Specific inhibitors of ERK, and phosphoinositide-3 kinase (PI3K), inhibited the HER-2 induced up-regulation of survivin. We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells.

Conclusion: Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells. We determined that survivin is up-regulated via ERK activation and PI3K signalling. Additionally we show that survivin up-regulation is not at transcriptional level. These data provide insight into the mechanism(s) by which induction of HER-2 over expression up-regulates survivin and Bcl-2 and identifies new targets for therapy of breast cancer.

Show MeSH
Related in: MedlinePlus