Article pubs.acs.org/JAFC
Demethoxycurcumin Modulates Prostate Cancer Cell Proliferation via AMPK-Induced Down-regulation of HSP70 and EGFR Chao-Ming Hung,† Yun-Hsuan Su,§ Hui-Yi Lin,⊗ Jia-Ni Lin,○ Liang-Chih Liu,△ Chi-Tang Ho,□ and Tzong-Der Way*,§,▽ †
Department of General Surgery, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan ⊗ School of Pharmacy, College of Medicine, College of Pharmacy, China Medical University, Taichung, Taiwan ○ Ph.D. Program for Cancer Biology and Drug Discovery, College of Pharmacy, China Medical University, Taichung, Taiwan △ Department of Surgery, China Medical University Hospital, Taichung, Taiwan □ Department of Food Science, Rutgers University, New Brunswick, New Jersey, United States ▽ Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan §
ABSTRACT: Curcumin (Cur), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are major forms of curcuminoids found in the rhizomes of turmeric. This study examined the effects of three curcuminoid analogues on prostate cancer cells. The results revealed that DMC demonstrated the most efficient cytotoxic effects on prostate cancer PC3 cells. DMC activated AMPK and in turn decreased the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). AICAR, an AMPK activator, and DMC down-regulated heat shock protein (HSP) 70 and increased the activity of the pro-apoptotic effector, caspase-3. In addition, DMC sustained epidermal growth factor receptor (EGFR) activation by suppressing the phosphatases PP2a and SHP-2. DMC also increased the interaction between EGFR and Cbl and induced the tyrosine phosphorylation of Cbl. The results suggest that DMC may have antitumor effects on prostate cancer cells via AMPK-induced down-regulation of HSP70 and EGFR. KEYWORDS: curcuminoids, demethoxycurcumin, AMPK, EGFR, HSP70
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metabolic disorders associated with increased cancer risk.6 Interestingly, a recent study suggests that AMPK dysregulation may provide a mechanistic link between metabolic syndrome and prostate cancer.7 Drugs that ameliorate metabolic syndrome conditions through AMPK activation may be beneficial for prostate cancer prevention and treatment. A family of highly conserved chaperone proteins, heat shock proteins (HSPs), is dramatically increased in response to heat stresses. Previous studies have indicated that HSP70 promotes tumorigenesis via its prosurvival function.8,9 High HSP70 expression, as observed in most cancer cells, has been associated with metastasis, poor prognosis, and resistance to chemotherapy or radiation therapy.10 Furthermore, HSP70 antisense constructs selectively kill cancer cells, not only in cell culture but also in various orthotopic tumor xenografts in mice.11−13 Thus, HSP70 seems to be an interesting molecular target for sensitizing tumor cells to cancer therapy. However, small molecules that are able to selectively inhibit HSP70 are still not known. Deregulations of epidermal growth factor receptor (EGFR) signaling are often associated with abnormal cell growth and survival in prostate cancer cells.14 Because ligand-dependent
INTRODUCTION Prostate cancer is a common urologic malignant tumor in men. It is the most common cancer, representing the second leading cause of cancer death.1 Chemotherapeutic treatment options for castrate-resistant prostate cancer have a very modest palliative and survival benefit. To improve survival in prostate cancer, new therapeutic strategies to inhibit the appearance of this phenotype must be developed. Emerging studies on cancer prevention and treatment with natural products expand the traditional treatment of prostate cancer. In contrast to normal human tissues, cancer cells display high rates of anabolic metabolism; overexpress lipogenic enzymes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN); and show a high rate of energy consumption driving increased protein synthesis2 and more active DNA synthesis.3 Recent studies have shown that inactivation of lipogenic enzymes, such as FASN, ACC, and 3-hydroxy-3methylglutaryl CoA reductase (HMG-CoA reductase), results in either cell death or growth inhibition in tumor cells.4,5 Nearly all prostate cancer cells express high levels of FASN, suggesting that metabolic pathways have become an attractive target for drug discovery against prostate cancer. AMP-activated protein kinase (AMPK), a highly conserved energy-sensing serine/threonine kinase, is activated by metabolic stress, for example, hypoxia, glucose deprivation, or exercise, which depletes intracellular ATP and increases AMP levels. Decreased AMPK activation is implicated in human © 2012 American Chemical Society
Received: Revised: Accepted: Published: 8427
June 28, 2012 July 31, 2012 July 31, 2012 July 31, 2012 dx.doi.org/10.1021/jf302754w | J. Agric. Food Chem. 2012, 60, 8427−8434
Journal of Agricultural and Food Chemistry
Article
Figure 1. Proliferation-inhibitory effect of curcuminoids on prostate cancer cells: (A) structures of curcumin (Cur), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC); (B) PC3 cells treated with various concentrations of Cur, DMC, and BMC at 37 °C for 48 h. The effect on cell growth was examined by MTT assay, and the percentage of cell proliferation was calculated by defining the absorption of cells without pterostilbene as 100%. This experiment was repeated three times. Bar represent the SEM. purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Cbl antibody was purchased from Upstate Technology (Lake Placid, NY, USA). Antibodies for mouse and rabbit conjugated with horseradish peroxidase (HRP) were purchased from Chemicon (Temecula, CA, USA). Western chemiluminescent HRP substrate was from Millipore Corp. (Billerica, MA, USA). Drug Preparation. The powdered roots from Curcuma longa (2.5 kg) were extracted with ethanol (EtOH) at room temperature. The EtOH extract (0.57 kg) was removed in vacuo at 40 °C and triturated with n-hexane, chloroform (CHCl3), and ethyl acetate (EtOAc) to give four fractions. The EtOAc-soluble fraction (20 g) was dissolved in methanol (MeOH) and then passed through a Sephadex LH-20 column, monitored by Si gel TLC analysis. A subfraction (2 g) was chromatographed on a Si gel column using a gradient elution (70 g, 70−230 mesh, 0−10% MeOH in CHCl3) to give three pigments, Cur, DMC, and BDMC (>98% purity). Cur, DMC, and BDMC were dissolved in dimethyl sulfoxide (DMSO) and diluted in RPMI-1640 medium to different final concentrations in the following experiments. Cell Culture. Human prostate cells and LNCaP, DU145, and PC3 cell lines were purchased from American Type Culture Collection. LNCaP, DU145, and PC3 cell lines were grown in RPMI-1640 media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin−streptomycin (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. MTT Assay. Cells (1 × 104) were seeded on the 24-well cell culture cluster overnight and then treated with different concentrations of agents as indicated in the figure captions and incubated for 48 h. Next, 40 μL of MTT (stock concentration = 2 mg/mL; Sigma Chemical Co.) was added to each well, and every volume of wells was 500 μL; this was followed by 2 h of incubation at 37 °C. MTT−formazan crystals will be formed; then 250 μL of DMSO was added to dissolve the crystals. Finally, an enzyme-linked immunosorbent assay (ELISA) reader was used to measure absorbance at OD 550 nm. Western Blot Analysis. Cells (1 × 106) were seeded onto a 100 mm tissue culture dish containing 10% FBS RPMI-1640. Cells were then treated with various agents as indicated in the figure captions. After treatment, cells were placed on ice, washed with cold PBS, and lysed in lysis buffer. Western blot analysis was done as described previously.25 Immunoprecipitation. Cells were lysed with TGH buffer [1% Triton X-100, 10% glycerol, 50 mM HEPES (pH 7.3), 50 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1
degradation of EGFR is a critical step for modulating receptor activity, targeting EGFR degradation may be an alternative approach to reduce cell signaling.15 EGFR is monoubiquitinated at multiple sites through the action of the E3 ubiquitin ligases casitas B-lineage lymphoma (Cbl). Cbl has a tyrosine kinase binding (TKB) domain and a RING finger E3 ubiquitin ligase domain that are required for targeted protein degradation.16 Cbl-mediated ubiquitination has been shown to play an important role during EGFR ubiquitination.17 However, the relationship between AMPK and EGFR ubiquitination is less clear. Curcuminoids are the major components that can be extracted from the rhizomes of Curcuma longa Linn.,18 and they consist of a mixture of curcumin (Cur, 75−80%), demethoxycurcumin (DMC, 15−20%), and bisdemethoxycurcumin (BDMC, 3−5%). Recent studies have shown that curcuminoids possess a wide spectrum of physiological activity. They display anticancer activity,19−21 promote neurite outgrowth,22 possess antimutagenic properties,23 and inibit influenza viruses.24 Currently, intense research efforts have focused on the potential of targeting metabolic pathways that may be altered during prostate tumorigenesis and prostate cancer progression. A significant amount of attention has been focused on the inhibition of tumor cell growth by the activation of AMPK. In the current study, we investigated the effects of curcuminoids on the viability of prostate cancer cells. Moreover, we plan to explore new therapeutic possibilities for the use of AMPK as an antiprostate tumor target.
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MATERIALS AND METHODS
Materials. Compound c and antibodies for β-actin were purchased form Sigma (St. Louis, MO, USA). 5-Aminoimidazole-4-carboxamide1-β-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals (Toronto, ON, Canada). Antibodies for FASN, phosphoACC (Ser 79), ACC, EGFR, phospho-EGFR, phospho-ERK1/2, ERK1/2, activated caspase-3, SHP-2, AMPK, and phospho-AMPK (Thr 172) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for HSP70 and tyrosine-specific antibody were 8428
dx.doi.org/10.1021/jf302754w | J. Agric. Food Chem. 2012, 60, 8427−8434
Journal of Agricultural and Food Chemistry
Article
Figure 2. DMC activates AMPK and its downstream targets, ACC and FASN. (A) PC3 cells were stimulated with DMSO (control) or different concentrations of DMC for 48 h. Cell lysates were analyzed by Western blot with anti-phospho-AMPK and anti-AMPK antibodies. (B) PC3 cells were stimulated with DMSO (control) or 20 μM DMC for the indicated times. Cell lysates were analyzed by Western blot with anti-phospho-AMPK and anti-AMPK antibodies. (C) PC3 cells were stimulated with DMSO (control) or different concentrations of DMC for 48 h. Cell lysates were analyzed by Western blot with anti-phospho-ACC, anti-ACC, and anti-FASN antibodies. (D) PC3 cells were stimulated with DMSO (control) or 20 μM DMC for the indicated times. Cell lysates were analyzed by Western blot with anti-phospho-ACC, anti-ACC, and anti-FASN antibodies. (E) DU145 and LNCaP cells were stimulated with DMSO (control) or 20 μM DMC for the indicated times. Cell lysates were analyzed by Western blot with anti-phospho-AMPK, anti-AMPK, anti-phospho-ACC, anti-ACC, and anti-FASN antibodies. Western blot data presented are representative of those obtained in at least three separate experiments. Immunoblots were quantified, and relative expression to control is indicated.
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mM phenylmethanesulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin]. The lysates were then centrifuged at 14000g for 10 min at 4 °C. The supernatants were incubated with anti-EGFR and anti-Cbl antibodies for 4 h and then washed with TGH buffer three times. The immunoprecipitates were subjected to sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Statistical Analysis. The results are expressed as the mean ± SEM. To analyze band intensity, Image Gauge (version 3.12, Fujifilm, Tokyo, Japan) was used. One-way ANOVA was used followed by Newman−Keuls multiple-range test to compare between groups. P values of
