Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells

Abstract

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.

Figure 1

Protein separation by two-dimensional gel electrophoresis. (A) Image of a Sypro Ruby-stained 2DE derived from 20 µg/ml GTE treated HPAF-II cell lysate. Cell lysate (100 µg) was isoelectric focused on 17-cm IPG (pH 3–10) strips followed by 8–16% SDS-PAGE. After Sypro Ruby staining, spot intensities were determined by Progenesis SameSpots 2-D Gel Analysis software. Proteins with enhanced or decreased expression levels in the presence of GTE compared to the control were selected, digested and identified by LC-MS/MS. The identities of numbered proteins on the gel are listed in Table 1. (B) Suppressed spots of Hsp90, Trap1 and Hsp27 by GTE treatment as compared to the control. (C) MS/MS spectra of tryptic peptide Q₈₀LSSGVSEIR₈₉ (2+ charge) from spot #12, showing the presence of both unphosphorylated peptide (top) and the phosphorylated peptide (bottom). The MS/MS spectrum indicates that Ser82 is phosphorylated (The peaks are labeled with conventional notation for the product ions. Labels marked with an asterisk denote the loss of 18 Da, or water, from the product ions).

Figure 1

Protein separation by two-dimensional gel electrophoresis. (A) Image of a Sypro Ruby-stained 2DE derived from 20 µg/ml GTE treated HPAF-II cell lysate. Cell lysate (100 µg) was isoelectric focused on 17-cm IPG (pH 3–10) strips followed by 8–16% SDS-PAGE. After Sypro Ruby staining, spot intensities were determined by Progenesis SameSpots 2-D Gel Analysis software. Proteins with enhanced or decreased expression levels in the presence of GTE compared to the control were selected, digested and identified by LC-MS/MS. The identities of numbered proteins on the gel are listed in Table 1. (B) Suppressed spots of Hsp90, Trap1 and Hsp27 by GTE treatment as compared to the control. (C) MS/MS spectra of tryptic peptide Q₈₀LSSGVSEIR₈₉ (2+ charge) from spot #12, showing the presence of both unphosphorylated peptide (top) and the phosphorylated peptide (bottom). The MS/MS spectrum indicates that Ser82 is phosphorylated (The peaks are labeled with conventional notation for the product ions. Labels marked with an asterisk denote the loss of 18 Da, or water, from the product ions).

Figure 1

Protein separation by two-dimensional gel electrophoresis. (A) Image of a Sypro Ruby-stained 2DE derived from 20 µg/ml GTE treated HPAF-II cell lysate. Cell lysate (100 µg) was isoelectric focused on 17-cm IPG (pH 3–10) strips followed by 8–16% SDS-PAGE. After Sypro Ruby staining, spot intensities were determined by Progenesis SameSpots 2-D Gel Analysis software. Proteins with enhanced or decreased expression levels in the presence of GTE compared to the control were selected, digested and identified by LC-MS/MS. The identities of numbered proteins on the gel are listed in Table 1. (B) Suppressed spots of Hsp90, Trap1 and Hsp27 by GTE treatment as compared to the control. (C) MS/MS spectra of tryptic peptide Q₈₀LSSGVSEIR₈₉ (2+ charge) from spot #12, showing the presence of both unphosphorylated peptide (top) and the phosphorylated peptide (bottom). The MS/MS spectrum indicates that Ser82 is phosphorylated (The peaks are labeled with conventional notation for the product ions. Labels marked with an asterisk denote the loss of 18 Da, or water, from the product ions).

Figure 2

Confirmation of heat shock proteins by Western blot analysis. HPAF-II cells were treated with 0, 20, and 40 µg/mL of GTE for 24 h. Data represent one of the two or three experiments.

Figure 3

GTE targeting Hsp90 client proteins and inducing apoptosis and growth suppression in HPAF-II cells. (A) Immunohistochemical analysis of HPAF-II cells treated with 0, 10, 20 and 40 µg/mL of GTE for 24 h. Cells were washed, fixed, and labeled sequentially for Hsp90, Akt or p53 (green), and (B) cleaved caspase-3 (green) and DNA (blue); and (C) effects of GTE on HPAF-II cell viability. Cells were plated at a density of 1×10⁴ cells/mL and treated with increasing concentrations of GTE for 24 and 48 h. Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Images were taken using a Nikon Eclipse 90i fluorescence microscope at ×20 objects. All data represent one of the two separate experiments, and three independent microscopic fields were examined for each sample.

Figure 3

GTE targeting Hsp90 client proteins and inducing apoptosis and growth suppression in HPAF-II cells. (A) Immunohistochemical analysis of HPAF-II cells treated with 0, 10, 20 and 40 µg/mL of GTE for 24 h. Cells were washed, fixed, and labeled sequentially for Hsp90, Akt or p53 (green), and (B) cleaved caspase-3 (green) and DNA (blue); and (C) effects of GTE on HPAF-II cell viability. Cells were plated at a density of 1×10⁴ cells/mL and treated with increasing concentrations of GTE for 24 and 48 h. Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Images were taken using a Nikon Eclipse 90i fluorescence microscope at ×20 objects. All data represent one of the two separate experiments, and three independent microscopic fields were examined for each sample.

Figure 3

GTE targeting Hsp90 client proteins and inducing apoptosis and growth suppression in HPAF-II cells. (A) Immunohistochemical analysis of HPAF-II cells treated with 0, 10, 20 and 40 µg/mL of GTE for 24 h. Cells were washed, fixed, and labeled sequentially for Hsp90, Akt or p53 (green), and (B) cleaved caspase-3 (green) and DNA (blue); and (C) effects of GTE on HPAF-II cell viability. Cells were plated at a density of 1×10⁴ cells/mL and treated with increasing concentrations of GTE for 24 and 48 h. Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Images were taken using a Nikon Eclipse 90i fluorescence microscope at ×20 objects. All data represent one of the two separate experiments, and three independent microscopic fields were examined for each sample.