Differential expression and regulation of prohibitin during curcumin-induced apoptosis of immortalized human epidermal HaCaT cells

Abstract

Prohibitin (PHB), also known as inhibin, is important in cell proliferation, differentiation and apoptosis. This protein localizes to the inner membrane of mitochondria, where it acts as a chaperone protein, and is also found in the nucleus, where it negatively regulates transcription. The tumor-suppressive role of PHB in cell proliferation appears to be contradictory. In this study, we investigated the existence, localization and alterations in the expression of PHB in the whole cell and nuclear matrix and analyzed its co-localization with the expression products of related genes. The western blot analysis results revealed that PHB exists in the composition of nuclear matrix proteins and that the expression level of PHB is significantly increased in the whole cell and markedly decreased in the nuclear matrix after curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment. The laser confocal scanning microscope results demonstrated the co-localization of PHB with p53, c-Myc, Bax, and Fas in HaCaT cells, and this co-localization region was transferred as a result of curcumin treatment. In addition, the results of the GST pull-down assay demonstrated the direct interaction of PHB with p53, c-Myc and Bax but not Fas in vitro. Results of the present study confirmed that the expression and distribution of PHB, which is a nuclear matrix protein, affect the apoptosis of HaCaT cells and its co-localization with specific gene products connected with cell apoptosis.

Figure 1

Expression of prohibitin (PHB) protein in HaCaT cells detected at whole cell and nuclear matrix proteins by western immunoblotting. Expression of PHB was found to be upregulated at whole cell and downregulated at nuclear matrix after curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment.

Figure 2

Effects of curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment on the localization of differentially expressed nuclear matrix proteins in the nuclear matrix-intermediate filament (NM-IF) system. (A) Light microscopy observation of NM-IF system in HaCaT cells, stained by Coomassie Brilliant Blue. (B) Light microscopy observation of the NM-IF system following curcumin treatment in HaCaT cells, stained by Coomassie Brilliant Blue. (C) Immunofluorescence staining of prohibitin (PHB) in HaCaT cells. (D) Immunofluorescence staining of PHB following curcumin treatment in HaCaT cells.

Figure 3

The co-localization between prohibitin (PHB) and c-Myc in HaCaT cells before and after treatment with curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). These results were observed by LSCM, PHB was labeled with FITC (green), c-Myc was labeled with TRITC (red). The co-localization fluorescence was yellow or orange when the two different colors overlapped.

Figure 4

The co-localization between prohibitin (PHB) and p53 in HaCaT cells before and after treatment with curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). These results were observed by LSCM, PHB was labeled with FITC (green) and p53 was labeled with TRITC (red). The co-localization fluorescence was yellow or orange when the two different colors overlapped.

Figure 5

The co-localization between prohibitin (PHB) and Fas in HaCaT cells before and after treatment with curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). These results were observed by LSCM, PHB was labeled with FITC (green) and Fas was labeled with TRITC (red). The co-localization fluorescence was yellow or orange when the two different colors overlapped.

Figure 6

The co-localization between prohibitin (PHB) and Bax in HaCaT cells before and after treatment with curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). These results were observed by LSCM, PHB was labeled with FITC (green) and Bax was labeled with TRITC (red). The co-localization fluorescence was yellow or orange when the two different colors overlapped.

Figure 7

Interactions between prohibitin (PHB) and the related tumor proteins (c-Myc, Bax and p53). GST pull-down assay was employed to verify the interaction between PHB and c-Myc, Bax and p53 in HaCaT cells.