Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50(Cdc37) complex and direct interactions with amino acids residues of Hsp90

Abstract

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with IC(50)s of around 10-15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50(Cdc37) in pancreatic cancer cells. Using nuclear magnetic resonance spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid chromatography coupled to mass spectrometry further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein-protein interaction in Hsp90 complex for its chemopreventive activity.

Fig. 1

Sulforaphane induces proteasomal degradation of Hsp90 client proteins. (a) Mia Paca-2 and Panc-1 cells were treated with 15 μM sulforaphane for different time periods. Sulforaphane induced a time-dependent down-regulation of Akt, Cdk4, and p53 mutant. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (b) Cells were treated with 5, 15, 25 μM sulforaphane. Sulforaphane induced a concentration-dependent down-regulation of these proteins. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (c) Cells were pre-incubated with 10 μM MG132 before sulforaphane treatment. Sulforaphane-induced down-regulation of Hsp90 client proteins were proteasome-mediated. SF, sulforaphane.

Fig. 1

Sulforaphane induces proteasomal degradation of Hsp90 client proteins. (a) Mia Paca-2 and Panc-1 cells were treated with 15 μM sulforaphane for different time periods. Sulforaphane induced a time-dependent down-regulation of Akt, Cdk4, and p53 mutant. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (b) Cells were treated with 5, 15, 25 μM sulforaphane. Sulforaphane induced a concentration-dependent down-regulation of these proteins. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (c) Cells were pre-incubated with 10 μM MG132 before sulforaphane treatment. Sulforaphane-induced down-regulation of Hsp90 client proteins were proteasome-mediated. SF, sulforaphane.

Fig. 1

Sulforaphane induces proteasomal degradation of Hsp90 client proteins. (a) Mia Paca-2 and Panc-1 cells were treated with 15 μM sulforaphane for different time periods. Sulforaphane induced a time-dependent down-regulation of Akt, Cdk4, and p53 mutant. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (b) Cells were treated with 5, 15, 25 μM sulforaphane. Sulforaphane induced a concentration-dependent down-regulation of these proteins. Densitometry data are presented as mean ± SD (n = 3, P < 0.05). (c) Cells were pre-incubated with 10 μM MG132 before sulforaphane treatment. Sulforaphane-induced down-regulation of Hsp90 client proteins were proteasome-mediated. SF, sulforaphane.

Fig. 2

Sulforaphane exhibits anticancer activity in vitro and in vivo. (a) Sulforaphane inhibited proliferation of Mia Paca-2, Panc-1, AsPc-1, and BxPc-3 cells. (b) Sulforaphane induced caspase-3 activity in Mia Paca-2 cells. Data are presented as means ± SD (n = 3, P < 0.01). (c) Anti-tumor effect of sulforaphane in xenografts. The pancreatic tumor xenograft model was generated by inoculating Mia Paca-2 cancer cells s.c. to the right and left flanks of nude mice. When the tumors reached 100-150 mm³, mice were randomly divided into three groups (n = 6) to receive vehicle, 25, or 50 mg/kg sulforaphane treatment (five times/week) for four weeks. Data are presented as means ± SD (n = 6, P < 0.01). (d) Mouse body weight was measured twice a week. SF, sulforaphane.

Fig. 2

Sulforaphane exhibits anticancer activity in vitro and in vivo. (a) Sulforaphane inhibited proliferation of Mia Paca-2, Panc-1, AsPc-1, and BxPc-3 cells. (b) Sulforaphane induced caspase-3 activity in Mia Paca-2 cells. Data are presented as means ± SD (n = 3, P < 0.01). (c) Anti-tumor effect of sulforaphane in xenografts. The pancreatic tumor xenograft model was generated by inoculating Mia Paca-2 cancer cells s.c. to the right and left flanks of nude mice. When the tumors reached 100-150 mm³, mice were randomly divided into three groups (n = 6) to receive vehicle, 25, or 50 mg/kg sulforaphane treatment (five times/week) for four weeks. Data are presented as means ± SD (n = 6, P < 0.01). (d) Mouse body weight was measured twice a week. SF, sulforaphane.

Fig. 3

Influence of sulforaphane on ATP binding of Hsp90 and Hsp90-cochaperone association in Mia Paca-2 cells. (a) Sulforaphane (5, 15, 30 μM) did not affect ATP binding, while 17-AAG (5 μM) decreased ATP binding to Hsp90. (b) Sulforaphane reduced the amount of p50^Cdc37 bound with Hsp90, while showed no effect on p23^Sba1. SF, sulforaphane.

Fig. 4

Sulforaphane binding to Hsp90 mapped by NMR. (a) Rainbow representation of ¹H-¹³C-Ile methyl TROSY cross peaks of full length Hsp90 bound to sulforaphane (1.5 mM) (blue to red gradient indicates increasing intensities; peak centers, black dots). (b) Overlayed peak position plots of Hsp90 Ile methyl TROSY spectra in absence (black) and presence of sulforaphane (orange), significant shifts are encircled (Combined chemical shift difference Δν = (0.25Δν_C² +Δν_H²)^1/2 > 0.01 ppm). (c) Homology model of human Hsp90 bound to p23^Sba1. (Hsp90 protomers, gray; p23^Sba1, light orange). The shifting pattern upon sulforaphane binding is indicated as space fillings in one monomer (chemical shifts Δν > 0.01 ppm, orange side chains; Δν > 0.015, red side chains; no shifts, blue δ-methyl groups; no assignments or inconclusive results, grey δ-methyl groups; lid of the ATP pocket, green backbone; cysteines, yellow). (d) Surface representation of Hsp90, zoomed into peptide stretch IDIIPNPQER (Hsp90, gray; IDIIPNPQER stretch, cyan; isoleucines that shift upon sulforaphane binding, red (Ile 75) and orange (Ile 74). (e) The Hsp90 N-terminal domain in complex with p50^Cdc37. (Hsp90, grey; p50^Cdc37, light orange; isoleucines and cysteines are colored as in (c).

Fig. 5

Proteolytic fingerprinting assay and LC-MS analysis of Hsp90 interaction with sulforaphane. (a) After incubation with DMSO or sulforaphane, protein sample (Hsp90βN) was digested with the indicated concentrations of trypsin. Hsp90 antibody (N-17), which detects N-terminus epitope of Hsp90, was used for immunoblotting. (b) Similarly, purified Hsp90βC protein was incubated with DMSO or sulforaphane, followed by trypsin digestion. The Hsp90 (AC88) antibody was used for immunoblotting. (c) Purified Hsp90βN was incubated with DMSO or sulforaphane (2 mM) for 30 min. Each sample was analyzed by LC-MS. (d) Purified Hsp90βN protein was incubated with DMSO or sulforaphane (2 mM) for 30 min. The samples were digested with Trypsin for 24 hrs at 37 °C and analyzed by LC-MS. SF, sulforaphane

Fig. 5

Proteolytic fingerprinting assay and LC-MS analysis of Hsp90 interaction with sulforaphane. (a) After incubation with DMSO or sulforaphane, protein sample (Hsp90βN) was digested with the indicated concentrations of trypsin. Hsp90 antibody (N-17), which detects N-terminus epitope of Hsp90, was used for immunoblotting. (b) Similarly, purified Hsp90βC protein was incubated with DMSO or sulforaphane, followed by trypsin digestion. The Hsp90 (AC88) antibody was used for immunoblotting. (c) Purified Hsp90βN was incubated with DMSO or sulforaphane (2 mM) for 30 min. Each sample was analyzed by LC-MS. (d) Purified Hsp90βN protein was incubated with DMSO or sulforaphane (2 mM) for 30 min. The samples were digested with Trypsin for 24 hrs at 37 °C and analyzed by LC-MS. SF, sulforaphane