Limits...
Circadian Rhythms and Breast Cancer: The Role of Per2 in Doxorubicin-Induced Cell Death.

Mitchell MI, Engelbrecht AM - J Toxicol (2015)

Bottom Line: Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen.Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities.Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Stellenbosch University, Stellenbosch, Matieland 7602, South Africa.

ABSTRACT
Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of various cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects on both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. This study was therefore aimed at characterizing the role of Per2 in normal breast epithelia (MCF-12A) and in ER(-) breast cancer cells (MDA-MB-231) and also at determining the role of Per2 in doxorubicin-induced cell death. In both cell lines Per2 protein expression displayed a 24-hour circadian rhythm in both cell lines. Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities. Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.

No MeSH data available.


Related in: MedlinePlus

Analysis of cell cycle progression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were subjected to (1) control conditions (C), (2) 2.5 μM doxorubicin (D), (3) 30 nM Per2 esiRNA (P), and (4) 30 nM Per2 for 48 hours + 2.5 μM Dox for 24 hours (PD). Cell cycle analysis was assessed following propidium iodide staining by flow cytometry. Statistical analysis: one way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3). P < 0.05 versus control and P < 0.001 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4548136&req=5

fig5: Analysis of cell cycle progression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were subjected to (1) control conditions (C), (2) 2.5 μM doxorubicin (D), (3) 30 nM Per2 esiRNA (P), and (4) 30 nM Per2 for 48 hours + 2.5 μM Dox for 24 hours (PD). Cell cycle analysis was assessed following propidium iodide staining by flow cytometry. Statistical analysis: one way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3). P < 0.05 versus control and P < 0.001 versus control.

Mentions: As cell cycle regulation is thought to be under the control of circadian genes, cell cycle analysis using flow cytometry was assessed to determine the effects of Per2 silencing on the ability of cells to progress through the cell cycle. Our results revealed that Per2 silencing alone leads to G0/G1 cell cycle arrest whereas Per2 silencing in combination with Dox leads to arrest in the S-phase of the cell cycle with a significant increase in apoptosis (Figure 5). Furthermore, G2/M transition analysis using conjugated primary antibodies against phosphorylated Histone H3 (Ser10) and phosphorylated Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) was assessed, in order to distinguish between cells in interphase, early mitosis, and late mitosis. Our results revealed a shift from early mitosis as well as a complete blunting of end stage mitosis with Dox treatment alone as well as in combination with Per2 silencing (Figure 6).


Circadian Rhythms and Breast Cancer: The Role of Per2 in Doxorubicin-Induced Cell Death.

Mitchell MI, Engelbrecht AM - J Toxicol (2015)

Analysis of cell cycle progression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were subjected to (1) control conditions (C), (2) 2.5 μM doxorubicin (D), (3) 30 nM Per2 esiRNA (P), and (4) 30 nM Per2 for 48 hours + 2.5 μM Dox for 24 hours (PD). Cell cycle analysis was assessed following propidium iodide staining by flow cytometry. Statistical analysis: one way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3). P < 0.05 versus control and P < 0.001 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4548136&req=5

fig5: Analysis of cell cycle progression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were subjected to (1) control conditions (C), (2) 2.5 μM doxorubicin (D), (3) 30 nM Per2 esiRNA (P), and (4) 30 nM Per2 for 48 hours + 2.5 μM Dox for 24 hours (PD). Cell cycle analysis was assessed following propidium iodide staining by flow cytometry. Statistical analysis: one way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3). P < 0.05 versus control and P < 0.001 versus control.
Mentions: As cell cycle regulation is thought to be under the control of circadian genes, cell cycle analysis using flow cytometry was assessed to determine the effects of Per2 silencing on the ability of cells to progress through the cell cycle. Our results revealed that Per2 silencing alone leads to G0/G1 cell cycle arrest whereas Per2 silencing in combination with Dox leads to arrest in the S-phase of the cell cycle with a significant increase in apoptosis (Figure 5). Furthermore, G2/M transition analysis using conjugated primary antibodies against phosphorylated Histone H3 (Ser10) and phosphorylated Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) was assessed, in order to distinguish between cells in interphase, early mitosis, and late mitosis. Our results revealed a shift from early mitosis as well as a complete blunting of end stage mitosis with Dox treatment alone as well as in combination with Per2 silencing (Figure 6).

Bottom Line: Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen.Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities.Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Stellenbosch University, Stellenbosch, Matieland 7602, South Africa.

ABSTRACT
Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of various cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects on both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. This study was therefore aimed at characterizing the role of Per2 in normal breast epithelia (MCF-12A) and in ER(-) breast cancer cells (MDA-MB-231) and also at determining the role of Per2 in doxorubicin-induced cell death. In both cell lines Per2 protein expression displayed a 24-hour circadian rhythm in both cell lines. Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities. Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.

No MeSH data available.


Related in: MedlinePlus