Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells

Experimental Eye Research 91 (2010) 211e219

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Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells Wen-Chuan Wu a,1, Meng-Hsien Wu a,1, Yo-Chen Chang a, b, Ming-Chu Hsieh a, Horng-Jiun Wu a, b, Kai-Chun Cheng a, b, Yu-Hung Lai a, b, **, Ying-Hsien Kao c, * a b c

Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, No 100, Zihyou 1st Rd., Kaohsiung 807, Taiwan Department of Ophthalmology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung 812, Taiwan Department of Medical Research, E-DA Hospital, I-Shou University, 6 E-DA Rd., Yan-Chau Shiang, Kaohsiung County 824, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2010 Accepted in revised form 7 May 2010 Available online 20 May 2010

Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with antifungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: geldanamycin 17-AAG Hsp70 Hsp90a cell cycle growth arrest ERK1/2 Akt

1. Introduction The molecular chaperone heat shock protein 90 (Hsp90) is constitutively expressed in mammalian cells and plays an essential role in facilitating proper folding, maturation, and activity of its client proteins in an ATP-dependent manner (Riggs et al., 2004). Once binding to the hydrophobic NH2 terminus pocket, ATP/ADP alters conformation of Hsp90 and results in its interaction with the cochaperone complex that protects or stabilizes the client proteins

* Corresponding author. Tel.: þ886 7 6151100x5059; fax: þ886 7 6155150. ** Corresponding author at: Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, No 100, Zihyou 1st Rd., Kaohsiung 807 Taiwan. Tel.: þ886 7 3214573; fax: þ886 7 3156413. E-mail addresses: [email protected] (Y.-H. Lai), [email protected] (Y.-H. Kao). 1 These authors equally contributed to this article. 0014-4835/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2010.05.005

(Grenert et al., 1997). Alternatively, Hsp90 directs the misfolded proteins, with a subset of cochaperones, to a covalent linkage with polyubiquitin and subsequent degradation by 26S proteasome (Riggs et al., 2004). Geldanamycin (GA, Fig. 1A), a benzoquinone ansamycin antibiotics originally isolated from Streptomyces hygroscopicus, possesses anti-fungal activity and inhibits plant pathogens (Heisey and Putnam, 1986). GA and its less toxic analog 17-allylamino-demethoxy geldanamycin (17-AAG, Fig. 1B), specifically bind to the ATP/ADP binding pocket of Hsp90, stabilize its conformation and prevent the recruitment of Hsp70-based cochaperone complex associated with the misfolded client proteins, thereby inhibiting Hsp90 function as a molecular chaperone. This action frequently results in ubiquitin-mediated proteasomal degradation of client proteins (Grenert et al., 1997; Isaacs et al., 2003). Hsp90 client proteins include the receptor c-Kit, the signaling proteins MEK, Raf-1, and Akt, and the cell cycle regulator cyclin D1, which are deregulated in cancers (Maloney and

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2.2. Cell culture Human primary RPE cells were isolated from a donor eye and the biochemical features were characterized using the method previously described (Chang et al., 2009; Wu et al., 2000). The cells were grown in the Ham’s F12 nutrient medium supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 4 mM glutamine (HyClone),100 I. U. penicillin per mL, 100 mg streptomycin per mL, and 250 ng amphotercin per mL (ICN Biomedicals, Inc., Aurora, Ohio) in a humidified atmosphere containing 5% CO2 at 37  C. Before experiment, the RPE cells were trypsinized, seeded in appropriate culturewares, and incubated overnight until attachment. Fig. 1. Structures of ansamycin antibiotics, GA (A) and 17-AAG (B).

2.3. Cell proliferation assay Workman, 2002; Sreedhar et al., 2004). Because treatment with GA causes depletion of a variety of oncogenic client proteins that are required for cell proliferation, survival, and metastatic angiogenesis (Mabjeesh et al., 2002; Sanderson et al., 2006), Hsp90 inhibitors clinically have attracted much attention as potential anti-cancer agents. The retinal pigment epithelium (RPE), lying between choriocapillary and neural retinal, is known to essentially support physiological function of adjacent photoreceptors. Emerging evidence indicated that RPE cell is one of the main component cell types in fibrous epi-retinal membrane observed in eyes with various vitreoretinal diseases, including proliferative vitreoretinopathy (PVR) (Jerdan et al., 1989; Machemer and Laqua, 1975). As PVR is in progress, the normally quiescent and differentiated RPE cells detach from the underlying Bruch’s membrane, migrate and proliferate (Kirchhof and Sorgente, 1989; Pastor, 1998). RPE cell proliferation is thus considered as a hallmark and, conversely, antiRPE cell proliferation may constitute one applicable therapeutic strategy for PVR. Although heat shock response has been noted in cultured RPE cells (Wakakura and Foulds, 1993), its pathophysiological role in ocular diseases is rarely discussed. Our previous study has demonstrated that GA treatment may suppress the hypoxiainduced VEGF production in cultured RPE cells (Wu et al., 2007), implicating its applicability in treating diseases with intraocular neovascularization. However, little is known about whether GA and/or its analogous chemical exhibit cytotoxicity on RPE cells. To evaluate the drug efficacy and safety, we treated cultured RPE cells with GA or 17-AAG, observed the drug-induced cytostatic and/or cytotoxic effects, and delineated the signaling scenario therein. 2. Materials and methods 2.1. Chemicals and antibodies GA and 17-AAG was purchased from AG Scientific (San Diego, CA). Ham’s F12 nutrient mixture (F12 medium), fetal bovine serum (FBS), glutamine, and trypsin-ethylene diamine tetraacetic acid (EDTA) were from Invitrogen Gibco (Gaithersburg, MD). The primary antibodies against Actin, p21, p53, phosphorp53 (serine 15), Hsp70 in Western blot analysis were from Santa Cruz Biotechnology (Santa Cruz, CA), those against total ERK1/2, phospho-EKR1/2 (threonine202/185 and tyrosine 204/187), total Akt, and phospho-Akt (serine 473) were from Invitrogen Biosource (Camarillo, CA). Antibody for Hsp90 alpha chain was from Millipore Chemicon (Temecular, CA). Secondary antibodies against mouse, rabbit, or goat IgG conjugated with horseradish peroxidase enzyme were from Jackson ImmunoResearch (West Grove, PA). Propidium iodide for cell cycle analysis was from Sigma (St. Luis, MO).

The cell-based proliferation assay was performed using commercially available MTS reagent (Promega, Madison, WI) as previously described (Wu et al., 2007). In brief, RPE cells, seeded in 96-well plates (5  103 cells/well) in triplicate, were performed with drug treatment. After incubation, MTS reagent was added and incubated for 1 h, followed by spectrophotometrical detection with microtiter plate reader (MRX II, Dynex technologies, Chantilly, VA). The proliferation was expressed as the percentage of control cells. 2.4. Cell cycle analysis by flow cytometry Cell cycle progression was monitored using DNA flow cytometry. In brief, after drug treatment, the cells were harvested by trpsinization, followed by fixation with 70% ethanol for 16 h at 4  C. The cells were then stained with 4 mg/ml of propidium iodide (PI) in PBS containing 1% Triton X-100 and 0.1 mg/ml of RNase A (Sigma) at 37  C for 1 h. The DNA content of individual cells was analyzed using a flow cytometer (EpicsÒXL/MCL, Beckman Coulter, Fullerton, CA), and the statistic cell cycle analysis was performed with WinCycle software (Phoenix Flow Systems), as described previously (Wu et al., 2003). 2.5. Apoptotic detection using annexin V and PI staining Annexin V-FITC Apoptosis Detection Kit (BioVision, CA) was used to detect both events of early apoptosis and necrosis induced by GA or 17-AAG. After 3 h of incubation at 10 mM, both adherent cells and floating cells were collected by trypsinization, centrifuged at 1000g for 5 min. Cell pellets were washed three times with PBS, resuspended in annexin V binding buffer with FITC-conjugated annexin V and incubated for 15 min in the dark. After incubation with PI for 5 min at room temperature, the dual fluorescent signals labeled was measured by using flow cytometer (Beckman Coulter) and analyzed by WinMDI software. Alternatively, the cells grown on chamber slides, after being treated with GA or 17-AAG, were directly stained with annexin V and PI for 5 min. Slides were mounted with buffered glycerol medium and the fluorescently visualized cells were observed under fluorescent microscope (IX70, Olympus Optical Co., Japan). 2.6. Western blotting Total cellular protein extracts from cultured RPE cells were obtained by lysing the cells in ice-cold RIPA buffer in the presence of a cocktail of protease inhibitors (Roche, Molecular Biochemicals, Mannheim, Germany) and phosphatase inhibitors (1 mM sodium fluoride and 1 mM sodium orthovanadate). After centrifugation, total protein was quantified using a bicinchonic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) and an equal amount of total protein for each lane was subjected to SDS-PAGE,

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using 8% acrylamide gels under reducing conditions. After electrophoresis, proteins were subsequently electrotransferred onto a PVDF membrane. Blots were then blocked in 5% skim milk in PBST at room temperature for 1 h, followed by overnight incubation at 4  C with primary antibodies. After five washes with PBS-T, the blots were incubated with secondary antibodies (HRP-conjugated anti-rabbit IgG) at 1:20 000 dilutions. The immunoreactive signals were visualized on x-ray film by enhanced chemiluminescence detection system (Millipore, Billerica, MA), documented on a digital imaging system (UVI Tech, Cambridge, UK) and analyzed in a densitometrical analysis system (UVI Tech). Relative protein levels were expressed as the ratios of densities between proteins and actin for each sample and normalized to that of negative control. 2.7. Statistical analysis The results are expressed as mean  standard deviation (SD). The unpaired Student’s t-test and ANOVA were used for statistical analysis. A level of p < 0.05 were considered to be a statistically significant difference. 3. Results 3.1. GA and its analog, 17-AAG, induce cytostaticity and cytotoxicity in cultured RPE cells To observe whether GA and its analog, 17-AAG, directly exhibit cytostatic or cytotoxic effect on growth of cultured RPE cells, a cellular viability assay was utilized to document the alteration in cell numbers based upon the activity of NADPH-dependent enzymes in intact cells. The MTS cell proliferation assay demonstrated that both GA and 17-AAG similarly elicited obvious cytostatic and cytotoxic effects on RPE cells. After 24 h treatment at 1 mM, RPE cell growth was significantly suppressed by 17-AAG only. Under exposure to the concentrations at 10 mM or higher, the RPE cell viability was reduced by GA and 17-AAG down to 80% and 65% of control levels, respectively (Fig. 2), whereas treatment with 0.1% of DMSO as equimolar solvent control does not affect the RPE cell viability (data not shown).

GA 17-AAG

Cell viability (% of control)

120 100

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80

* ** * *

** ** **

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3.2. GA and 17-AAG induce both growth arrest and cell death of cultured RPE cells To determine whether higher concentrations of GA or 17-AAG induce cytostatic and/or cytotoxic effects on RPE cells, the cultured cells under exposure to either drug were subjected to cell cycle analysis and concomitant measurement for sub-G1 DNA content using flow cytometry. The results in Table 1 indicate that both GA and 17-AAG at concentrations higher than 1 mM exhibited similar apoptosis-inducing effect in terms of the appearance of cell population in sub-G1 DNA content area. Besides, both GA and 17-AAG at 1 mM or higher prominently increased G0/G1 subpopulation and, conversely, decreased G2/M subpopulation. Moreover, a remarkable increase in S phase population was noted in cells treated with either drug at 10 mM, while cells in G2/M phase were not detectable. These findings suggest that both drugs may induce RPE cell growth arrests in both G1 and S phases of cell cycle, as well as simultaneous RPE cell death. Next, to discriminate the pattern of ansamycininduced RPE cell death, the cells treated were detected by using a sensitive Annexin V-FITC apoptosis detection kit, followed flow cytometry. The results clearly indicated that GA and 17-AAG treatment for 12 h significantly induced both early and late apoptosis of RPE cells, as evidenced by flow cytometrical quantification (Fig. 3A) as well as by fluorescent microscopic observation (Fig. 3B). 3.3. GA and 17-AAG upregulate p53 and p21 expression To observe whether p53 and p21 pathway is involved in GA- or 17-AAG-elicited RPE cell death, the cells treated with either GA or 17-AAG were collected at different time points and subjected for Western blotting and subsequent densitometrical analyses (Fig. 4). The time course observation clearly indicated that total p53 contents in cells insulted by GA and 17-AAG remarkably elevated after 24 h and 12 h of treatment, respectively (Fig. 4A and B). In parallel, the increase in p53 phosphorylation at serine 15 was noted earlier than the alteration in total p53 content (Fig. 4A and C). In addition, the protein abundance of p21 downstream of p53 was remarkably detected in both cases, while the p21 induction by 17-AAG appeared more potent than that by GA (Fig. 4A and D). To determine whether GA and 17-AAG pharmacologically modulate expression of apoptotic regulatory proteins in cultured RPE cells, the cells treated with either GA or 17-AAG were collected after 24 h of treatment and subjected for Western blotting and subsequent densitometrical analyses. The results clearly indicated that both GA and 17-AAG remarkably upregulated expression of Hsp70 and Bcl-2 protein, while they downregulated Bax expression on the contrary (Fig. 5). Collectively, GA and 17-AAG treatment significantly increased both Hsp70-to-Bax and Bcl-2-to-Bax ratios.

60 Table 1 Effect of GA and 17-AAG on the cell cycle distribution of human RPE cells.

40 20 0

0

0.01

0.1

1

10

20

μM) Ansamycins (μ Fig. 2. Effects of GA and 17-AAG on viability of cultured human RPE cell. Cultured human RPE cells were seeded onto a 96-well plate at 24 h before drug treatment. A cell-based proliferation assay (MTS reagent) was performed after treatment with either GA or 17-AAG for 24 h. Data are representative results in three independent experiments, and expressed as mean  SD. ** and *** indicate P < 0.01 P < 0.001, respectively, compared with negative controls.

Experimental Groups

Cell cycle distribution

NC GA (0.1 mM) GA (1 mM) GA (10 mM) 17-AAG (0.1 mM) 17-AAG (1 mM) 17-AAG (10 mM)

4.8 5.2 12.4 14.3 3.1 11.0 13.7

% Sub-G1       

1.8 0.5 1.8* 2.0* 0.5 1.3* 2.6*

% G0/G1 62.4 63.1 70.7 69.2 63.8 69.4 70.3

      

2.7 0.4 2.0* 2.9* 1.3 1.5* 1.6*

%S 13.9 15.0 13.0 16.5 15.5 17.5 16.8

% G2/M       

2.0 0.9 1.7 1.8* 1.8 1.2* 1.5*

18.7 16.7 3.9 ND 17.6 2.1 ND

 2.1  3.6  0.9*  1.8  0.6*

Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment and treated with either GA or 17-AAG at indicated concentrations for 24 h. Cellular DNA content was determined by PI staining and subsequent flow cytometry. Data are shown as mean  SD of three independent experimental results. * annotates P < 0.05, compared with negative control (NC). ND, not detectable.

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Fig. 3. Apoptotic induction of cultured human RPE cells by GA and 17-AAG treatment. (A) The cells treated with GA or 17-AAG at 10 mM for 12 h were collected for staining with FITC-conjugated annexin V (FL1) and PI stain (FL2), followed by subsequent flow cytometrical measurement. The representative dot plot using FL1-FL2 parameters was analyzed in a quadrant mode. Percentage (%) of cell population was shown in each quadrant. (B) The GA- or 17-AAG-treated adherent cells were directly stained with annexin V and PI, then the fluorescently visualized cells were documented through fluorescent microscope. The early apoptotic events induced by ansamycins were evidenced by membrane staining pattern (indicated by arrows) of annexin V-FITC (middle panels), whereas the late apoptotic events by both membraneous and PI signals (right). Negative control (NC), cells without ansamycin treatment (left). Bar, 100 mm.

3.4. GA and 17-AAG modulate Hsp70 and Hsp90a expression To confirm the inductive effect of GA or 17-AAG on Hsp70 and Hsp90a subunit expression in cultured RPE cells, the protein levels of both proteins were measured by Western blotting detection (Fig. 6A). The densitometrical results showed that both GA and 17AAG significantly upregulated protein expression levels of Hsp70 (Fig. 6B) and Hsp90a (Fig. 6C) immediately at 3 h after drug treatment. Besides, the maximal induction of Hsp90a protein by GA was at 12 h, whereas that by 17-AAG was at 24 h after addition of drug. 3.5. GA and 17-AAG suppress ERK1/2 and Akt signaling activities To scrutinize the modulatory effects of GA and 17-AAG on survival signaling pathways in cultured RPE cells, the total protein contents and phosphorylation status of ERK1/2 and Akt were measured by Western blotting (Fig. 7). The results demonstrate that constitutive ERK1/2 phosphorylation was significantly reduced at 3 h and dramatically silenced at 6 h after treatment with GA and

17-AAG (Fig. 7A and B). Interestingly, the total ERK1/2 contents were notably reduced by GA, but not by 17-AAG treatment (Fig. 7A and C). In contrast, Akt phosphorylation was maximally and transiently induced at 3 h or earlier, then rapidly disappeared after 6 h after addition of drugs (Fig. 7A and D). Furthermore, constitutive levels of total Akt protein were remarkably reduced by both GA and 17-AAG (Fig. 7A and E). 3.6. GA and 17-AAG attenuate wortmannin-sensitized Akt phosphorylation Since a pilot study demonstrated that wortmannin, a selective inhibitor for phosphatidylinositol 3-kinase (PI3K), induced Akt phosphorylation in cultured human RPE cells (data not shown), we tested whether GA and 17-AAG could also suppress the wortmanninsensitized Akt activation. The cultured human RPE cells grown in normal culture medium with 10% FBS were pretreated with 10 mM wortmannin for 30 min, followed by treatment with GA or 17-AAG. Western blotting detection revealed that Akt phosphorylation

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Fig. 4. Induction of p53 phosphorylation and p21 upregulation in cultured human RPE cells by GA and 17-AAG treatment. Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment and followed by either 10 mM of GA or 17-AAG for indicated periods. Total protein was extracted and then subjected to Western blotting detection. (A) Representative photographs for cellular contents of total P53, phosphorylated P53 and P21 proteins. Quantification of relative total p53 (B), phosphorylated p53 (C), and p21 contents (D) was performed using pixel density analysis and is shown as induction folds of negative control. Values are expressed as mean  SD from three independent experiments. * indicates P < 0.05, compared with corresponding negative control.

was reproducibly seen in the DMSO-treated cells after 6.5 h of wortmannin treatment. However, the wortmannin-elicited Akt phosphorylation was completely abrogated by addition of GA or 17-AAG (Fig. 8A and B). This finding confirmed that GA and 17-AAG not only reduced constitutive abundance of Akt protein, but also potently inhibited wortmannin-sensitive Akt signaling pathway, suggesting its applicability in ceasing Akt-dependent cell behaviors including proliferation. 4. Discussion The benzoquinone ansamycin antibiotics GA and its analog 17AAG specifically bind to the ATP/ADP binding pocket of Hsp90, stabilize its conformation and prevent recruitment of misfolded client proteins, thereby disrupting its chaperone function for several transcription factors and protein kinases (Blagosklonny et al., 1995; Smith et al., 1998; Stancato et al., 1997). They have been currently suggested to be an effective therapeutic agent for suppressing cancer cell growth (Mabjeesh et al., 2002; Sanderson et al., 2006). In this study, we demonstrated that both GA and 17-AAG generate cytostaticity and cytotoxicity in cultured human RPE cells. The regulatory role of GA in cell cycle progression effects mainly lies in its modulatory function in triggering p53-p21 signaling activity (Lin et al., 2008; McIlwrath et al., 1996), thereby effectively inducing RPE cell growth arrest at lower concentrations or even apoptosis at higher concentrations. The latter event could be evidenced by emergence of sub-G1 hypodiploid cells (Table 1) as well as Annexin V-positive cells (Fig. 3). In addition, GA and 17-AAG

shifts the equilibrium between Hsp70 and Bax and that between Bcl-2 and Bax, suggesting its impact on apoptosis-related signaling. Moreover, both drugs not only effectively diminished ERK1/2 phosphorylation, but also silenced intracellular Akt contents. The attenuating effect was also seen in the event of wortmanninsensitized Akt phosphorylation. These data show for the first time that GA and 17-AAG mitigate Akt signaling pathway in RPE cells and reveal a mechanistic basis of GA- and 17-AAG-induced cytostaticity and cytotoxicity therein, thus strongly supports their possible application on anti-proliferation of RPE cells in PVR patients. Indeed, ansamycins have been shown to impact growth by inhibiting cell division in a variety of cancer cells such as glioblastomas (Garcia-Morales et al., 2007; Nomura et al., 2007), cervical cancer (Bisht et al., 2003), leukemia (Sugimoto et al., 2008), and hepatocellular carcinoma (Pritchard et al., 2009). The effective doses for inhibition of cancer cell growth largely range from submicromolar level when singly used for longer than 24 h, to less than 100 nM when combined with other modalities. As expected, this study evidenced that both GA and 17-AAG suppressed RPE cell growth at concentrations above 1 mM when treated for 24 h (Fig. 2). The effective dose range was generally similar to that seen in tumor cells, in spite of different duration of drug treatment. Several lines of evidence demonstrated that ansamycin treatment increased the percentage of cells arrested in both G1 and G2/M phases (Shelton et al., 2009), while the present study found that GA and 17-AAG elicited both G1 and S phase arrest (Table 1). The ansamycininduced G1 growth arrest is consistent with that seen in the leukemia cells carrying wild-type p53 (Lin et al., 2008). Taken

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Fig. 5. Alteration in expression of apoptosis-regulatory proteins in cultured human RPE cells by GA and 17-AAG treatment. Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment and followed by either GA or 17-AAG at indicated dose for 24 h. Total protein was extracted and then subjected to Western blotting detection. (A) Representative photographs for cellular contents of Hsp70, Bcl-2, and Bax proteins. (B) Quantification was performed using pixel density analysis and is shown as induction folds of negative control. Values are expressed as mean  SD from three independent experiments. * indicates P < 0.05, compared with corresponding negative control (NC).

together with the data showing a correlation between the expression patterns of p53 and p21 (Fig. 4), these findings strongly suggest that ansamycins induce G1 arrest in cultured RPE cells in a p53-dependent fashion. However, we still cannot rule out the possibility that ansamycins modulate the other p53-irrelevant signaling, because GA also induces p21 upregulation through intervening in p53/ATM-independent pathway (Lin et al., 2008; McIlwrath et al., 1996). Furthermore, p21 upregulation has been known to block kinase activity of cdc2 protein, a key kinase for G2/M checkpoint (Taylor and Stark, 2001). In fact, a p53-dependent cdc2 downregulation was previously reported in cells with ionizing radiation (Azzam et al., 1997). Moreover, the ansamycin-arrested tumor cell growth has been recently demonstrated to be achieved through decreasing cdc2 via protein degradation (Garcia-Morales et al., 2007; Nomura et al., 2007; Shelton et al., 2009; Watanabe et al., 2009). These findings, collectively, support the involvement of cdc2 activity in GA-elicited G1 arrest in cultured RPE cells. However, the exact mechanistic role of p53 in regulating cdc2

Relative Hsp90 α expression (induction folds)

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B Relative Hsp70 expression (induction folds)

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20 18 16 14 12 10 8 6 4 2 0

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Time (hrs) Fig. 6. Effect of GA and 17-AAG on heat shock protein induction in cultured human RPE cells. Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment and followed by either GA or 17-AAG at 10 mM dose for indicated time periods. Total protein was extracted and then subjected to Western blotting detection. (A) Representative photographs for cellular contents of Hsp70 and Hsp90a proteins are shown. Quantification of relative Hsp70 (B) and Hsp90a (C) expression was performed using pixel density analysis and is shown as induction folds of negative control. Values are expressed as mean  SD from three independent experiments. * indicates P < 0.05, compared with corresponding negative control.

kinase activity and cell cycle progression in cultured RPE cells remains to be further clarified. It is well known that equilibrium between Bcl-2 and Bax may reflect the anti-apoptotic propensity of cells (Del Poeta et al., 2003). Besides, high level of Hsp70 has been shown to inhibit the intrinsic apoptotic pathway by preventing the recruitment of procaspase-9 to Apaf-1 apoptosome. Similarly, ratio of Hsp70-toBax has been considered, to certain degree, as an anti-apoptotic indicator (Kao et al., 2008; Yoon et al., 2002). In this study, we attempted to present the pro-apoptotic tendency of GA and 17-AAG treatment in ratios of Bcl-2/Bax and Hsp70/Bax. A good correlation was seen between the ratios of Bcl-2/Bax and Hsp70/

W.-C. Wu et al. / Experimental Eye Research 91 (2010) 211e219

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Fig. 7. Effects of GA and 17-AAG on Erk1/2 and Akt activities in cultured human RPE cells. Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment and followed by either GA or 17-AAG at 10 mM dose for indicated time periods. Total protein was extracted and then subjected to Western blotting detection. (A) Representative photographs for cellular contents of total and phosphorylated ERK1/2 and Akt are shown. Quantification of phosphorylated ERK1/2 (B) and Akt (D) levels as well as contents of total ERK1/2 (C) and Akt (D) was performed using pixel density analysis and is shown as induction folds of negative control. Values are expressed as mean  SD from three independent experiments. * indicates P < 0.05, compared with corresponding negative control.

Bax, but an inconsistency was unexpectedly found between the trends of both ratios and the substantial drug effect (Fig. 5). The controversy is mainly due to the fact that both GA and 17-AAG induce prominent upregulation of Hsp70 and Bcl-2. A possible explanation for this contradictory phenomenon may be the depletion of intracellular Akt protein induced by GA or 17-AAG treatment (Fig. 7). In fact, the ability of 17-AAG to downregulate key survival signaling protein kinases, such as c-Raf-1, c-Src, and Akt, has been previously demonstrated (Lin et al., 2008; Nimmanapalli et al., 2003). Besides, Akt activity is reported to play a pivotal role in regulation of Bcl-2 phosphorylation, thereby modulating the threshold of programmed cell death (Asnaghi et al., 2004; Tran et al., 2002). The upregulated Bcl-2 seen in

GA- or 17-AAG-treated cells, thus, might not function in terms of anti-apoptosis. With regard to the suppressive effect of GA and 17-AAG on the constitutive high ERK1/2 activity shown in cultured RPE cells, B-Raf/MEK/ERK signaling pathway is recently demonstrated to be targeted by 17-AAG for cell proliferation through the control of B-Raf expression (Babchia et al., 2008). In line with our observation, the significance of inhibition of Akt and ERK1/2 function by 17-AAG was previously reported in glioblastoma cells (Yacoub et al., 2008). Our findings highlight the superior importance of reduced constitutive levels and dephosphorylation of Akt and ERK1/2 in the ansamycin-induced cytostaticity and cytotoxicity on cultured RPE cells. Taken together with our in vitro findings, we propose that ansamycins may interrupt the signaling

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Time (hrs) after adding ansamycins Fig. 8. Effects of GA and 17-AAG on wortmannin-sensitive Akt phosphorylation in cultured human RPE cells. Cultured human RPE cells were seeded onto dishes at 24 h before drug treatment. The cells grown in normal culture medium were pretreated with 10 mM wortmannin (WM) for 30 min, and followed by addition of either 10 mM GA, 17-AAG or 0.1% DMSO as solvent control. At indicated time point, cellular lysates were extracted and then subjected to Western blotting detection. (A) Representative photographs for cellular contents of total and phosphorylated Akt proteins. (B) Quantification was performed using pixel density analysis and is shown in arbitrary unit. Values are expressed as mean  SD from three independent experiments. * indicate P < 0.05, compared with solvent control.

pathways of not only Akt-mediated survival but also Raf/MEK/ ERK-regulated proliferation of human RPE cells (Fig. 9). However, the mechanistic details in ansamycin-mediated signaling in RPE cells remain to be determined. The induction of Hsp70 by various cellular stresses, such as heat shock, heavy metal ions, oxidative radicals, and anti-cancer agents is functionally to prevent protein aggregation and facilitate protein refolding. Experimental evidence identifies abundantly expressed Hsp70 in malignant tumors as a cancer survival protein that increases cell proliferation, metastasis, and poor therapeutic outcome in human cancers. The GA-inducible Hsp70 and Hsp90 in cultured RPE cells is not only noted in this study, but has been previously reported to attenuate cytotoxicity of protein phosphatase inhibitor (Kaarniranta et al., 2005). Previous mechanistic study has demonstrated that GA or 17-AAG disrupts the association between Hsp90 and heat shock factor 1 (HSF-1), thereby promoting nuclear localization, heat shock element binding, and activity of HSF-1, resulting in induction of Hsp70 levels. More recently, GA-induced Hsp70 expression was found to exhibit anti-fibrotic effect through suppressing transforming growth factor-b (TGF-b) signaling (Yun et al., 2010). GA treatment effectively mitigated

Fig. 9. A schematic diagram representing proposed mechanism by which ansamycins intervene in Ark- and ERK1/2-mediated RPE cell proliferation. Based on previous findings in the literature (dashed lines) and our observations in the present study (solid lines), the diagram outlines the proposed pharmacological intervention of ansamycin antibiotics, including geldanamycin (GA) and 17-allylamino-demethoxy geldanamycin (17-AAG), in the PI3K/Akt-mediated survival and Raf/MEK/ERK1/2mediated proliferation signaling pathways in human RPE cells. Akt mediates downstream Bcl-2 phosphorylation responsible for cell survival by anti-apoptosis, while activated ERK1/2 regulates cyclin D1 phosphorylation and subsequent G1-S transition in cell cycle progression, thereby stimulating cell proliferation. This study evidenced that GA and 17-AAG exhibit RPE cytostaticity as well as cytotoxicity by virtue of downregulating constitutive expression levels of Akt and ERK1/2 proteins and dramatically by diminishing phosphorylation of both Akt and ERK1/2. Abbreviations: PI3K, phosphoinositide 3-kinase; Hsp90, heat shock protein 90; Raf, c-Raf kinase protein; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase; P, phosphorylated group.

TGF-b-induced Smad3 phosphorylation in tumor cells and induced degradation of TGF-b type I and type II receptors through a proteasome-dependent pathway. Since dysregulation of TGF-b signaling has long been implicated in the pathogenesis of PVR (Hinton et al., 2002; Limb et al., 1991), therefore we proposed that GA or 17-AAG may be potentially used for treating PVR, through not only anti-proliferative but also anti-fibrogenic effects, although the precise mechanism remains to be deciphered. In summary, the present study delineates the pathophysiological role of Hsp90 in cultured RPE cells. Our results provide the first evidence clearly showing that ansamycins exhibits both cytostaticity and cytotoxicity in RPE cells, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. However, it still remains obscure how ansamycins intervene cell cycle progression of RPE cells. Further study may refine definition of the signals required to modulate RPE cell proliferation, which can fortify the rationale for its possible application in PVR treatment. Acknowledgements This study was supported in part by the grants from the National Science Council, Taiwan (NSC97-2314-B-037-022) and from Kaohsiung Medical University Research Project (KMUH97-7R-10). References Asnaghi, L., Calastretti, A., Bevilacqua, A., D’Agnano, I., Gatti, G., Canti, G., Delia, D., Capaccioli, S., Nicolin, A., 2004. Bcl-2 phosphorylation and apoptosis activated

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