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A Novel Hsp90 Inhibitor to Disrupt Hsp90/Cdc37 Complex Against Pancreatic Cancer Cells

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A Novel Hsp90 Inhibitor to Disrupt Hsp90/Cdc37 Complex Against Pancreatic Cancer Cells 162 A novel Hsp90 inhibitor to disrupt Hsp90/Cdc37 complex against pancreatic cancer cells Tao Zhang,1 Adel Hamza,2 Xianhua Cao,1 Introduction 1 1 2 Bing Wang, Shuwen Yu, Chang-Guo Zhan, Pancreatic cancer is the fourth leading cause of cancer and Duxin Sun1 death in the United States (1). The overall 5-year survival 1 rate for pancreatic cancer patients is only 4% due to the lack Division of Pharmaceutics, College of Pharmacy, The Ohio State of treatment options (2). Currently, targeted therapy University, Columbus, Ohio and 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, against a malfunctioning molecule or pathway has pro- Lexington, Kentucky duced promising results for various solid tumors, such as Gefitinib against lung cancer, Avastin and Ebitux against colon cancer, Herceptin against breast cancer, and SU- Abstract 011248 against kidney cancer (3, 4). However, clinical trials Pancreatic cancer is an aggressive disease with multiple using antibodies against epidermal growth factor receptor, biochemical and genetic alterations. Thus, a single agent vascular endothelial growth factor, and HER-2 in pancre- to hit one molecular target may not be sufficient to treat atic cancer have had minimal benefits (5–7). Considering this disease. The purpose of this study is to identify a the complexity of pancreatic cancer with its multiple novel Hsp90 inhibitor to disrupt protein-protein interac- genetic abnormalities, targeting a single pathway is tions of Hsp90 and its cochaperones for down-regulating unlikely to be effective. Thus, identification of new targets many oncogenes simultaneously against pancreatic that modulate multiple signaling pathways would be cancer cells. Here, we reported that celastrol disrupted desired for treating pancreatic cancer. Hsp90-Cdc37 interaction in the superchaperone complex Therefore, targeting molecular chaperone Hsp90 may to exhibit antitumor activity in vitro and in vivo. offer many advantages for pancreatic cancer therapy due to Molecular docking and molecular dynamic simulations its simultaneous regulation of various oncogenic proteins showed that celastrol blocked the critical interaction of (8, 9). Most of Hsp90 client proteins (HER-2/neu, epidermal Glu33 (Hsp90) and Arg167 (Cdc37). Immunoprecipitation growth factor receptor, Akt, Cdk4, Bcr-Abl, p53, and Raf-1) confirmed that celastrol (10 Mmol/L) disrupted the are essential for cancer cell survival and proliferation (9). Hsp90-Cdc37 interaction in the pancreatic cancer cell Chaperoning of these client proteins is regulated through a line Panc-1. In contrast to classic Hsp90 inhibitor dynamic cycle driven by ATP binding to Hsp90 and (geldanamycin), celastrol (0.1-100 Mmol/L) did not subsequent hydrolysis (10). Hsp90 requires a series of interfere with ATP binding to Hsp90. However, celastrol cochaperones to forma complexfor its function. These (1-5 Mmol/L) induced Hsp90 client protein degradation cochaperones, including Cdc37, Hsp70, Hsp40, Hop, Hip, (Cdk4 and Akt) by 70% to 80% and increased Hsp70 p23, pp5, and immunophilins, bind and release in the expression by 12-fold. Celastrol induced apoptosis superchaperone complex at various times to regulate the in vitro and significantly inhibited tumor growth in folding, assembly, and maturation of Hsp90 client proteins Panc-1 xenografts. Moreover, celastrol (3 mg/kg) effec- (10). A well-known mechanism of Hsp90 inhibition tively suppressed tumor metastasis by more than 80% involves the compounds geldanamycin and its derivative in RIP1-Tag2 transgenic mouse model with pancreatic 17-allyamino-geldanamycin blocking ATP binding to islet cell carcinogenesis. The data suggest that celastrol Hsp90. These compounds cause the catalytic cycle of is a novel Hsp90 inhibitor to disrupt Hsp90-Cdc37 Hsp90 to arrest in the ADP-bound conformation, subse- interaction against pancreatic cancer cells. [Mol Cancer quently leading to premature release and degradation of Ther 2008;7(1):162–70] client proteins (10). This method has proven to be feasible therapeutically, such that 17-allyamino-geldanamycin has entered clinical trials (11). However, all current Hsp90 inhibitors employ the same mechanism of ATP blockage for inactivating this chaper- one. None of these inhibitors has received the Food and Received 7/18/07; revised 10/23/07; accepted 11/28/07. Drug Administration approval. It would be premature to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked conclude that the strategy of blocking the ATP binding to advertisement in accordance with 18 U.S.C. Section 1734 solely to Hsp90 is a viable approach for the development of a Hsp90 indicate this fact. inhibitor. In addition, many compounds that might have Requests for reprints: Duxin Sun, Division of Pharmaceutics, College of inhibited the function of Hsp90 were probably excluded Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210. Phone: 614-292-4381; Fax: 614-292-7766. during drug screening simply because they could not bind E-mail: [email protected] to the ATP pocket. Because the Hsp90 chaperoning process Copyright C 2008 American Association for Cancer Research. involves the transient formation of multiprotein complexes doi:10.1158/1535-7163.MCT-07-0484 with cochaperones, halting the chaperoning cycle at MolCancerTher2008;7(1).January2008 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 163 various stages is also likely to achieve Hsp90 inhibition. of islet tumors in vivo. These data provide a novel strategy Thus, we hypothesize that disruption of the interaction of for Hsp90 inhibition through disruption of Hsp90-cocha- Hsp90 with its cochaperones will achieve Hsp90 inhibition perones interaction, offering the potential of isolating against cancer cells. Hsp90 inhibitors against pancreatic cancers. Because one of the essential cochaperones is Cdc37, which associates client proteins to Hsp90, we intend to identify a Hsp90 inhibitor that disrupts the Hsp90-Cdc37 Materials and Methods interaction against pancreatic cancers in the present study. Drugs and Reagents We hypothesize that disruption of Hsp90-Cdc37 interac- Celastrol was purchased from Cayman Chemical, pro- tion, without affecting ATP binding to Hsp90, should have teasome inhibitor MG132 from Calbiochem, and lactacystin similar anticancer effects as treatment with 17-allyamino- from Sigma-Aldrich. Geldanamycin was kindly provided geldanamycin and geldanamycin. Previous studies have by Dr. George Wang (Department of Chemistry, The Ohio shown that celastrol (Fig. 1A), a quinone methide triterpene State University). The following antibodies were used for from Tripterygium wilfordii Hook F regulated the Hsp90 immunoblotting: Akt, Hsp90 (Cell Signaling), Hsp70, Hop pathway and induced the heat shock response similar to (StressGen), Cdk4, Cdc37, InBa, p27, Hsp90 (Santa Cruz), other Hsp90 inhibitors (12, 13). We found that celastrol did actin, and p23 (Abcam). Monoclonal Hsp90 antibody H9010 not effect ATP binding to Hsp90. Using molecular and purified Hsp90h protein were kind gifts of Dr. David modeling, we identified that celastrol blocked Cdc37 Toft (Mayo Clinic). binding to Hsp90. Indeed, immunoprecipitation confirmed Molecular Modeling that celastrol disrupted the Hsp90-Cdc37 complex. This The initial coordinates of the human Hsp90 protein used disruption resulted in degradation of Hsp90 client proteins in our computational studies came from the X-ray crystal to induce apoptosis in pancreatic cancer cell line Panc-1 structure (1US7.pdb; ref. 14) deposited in the Protein Data in vitro. In addition, celastrol exhibited anticancer activity Bank (15). For comparison, the yeast Hsp90-p23/Sba1 in Panc-1 xenograft model and RIP1-Tag2 transgenic mice X-ray structure 2CG9.pdb was also used (16). Using these Figure 1. Molecular dockingof celastrol with Hsp90 and Hsp90-Cdc37 complex. A, chemical structure of celastrol. B, ribbon view of the Hsp90-Cdc37 X-ray structure with the solvent accessible surface of the Hsp90-Cdc37 interface. C, ribbon view and solvent accessible surface of the Hsp90-celastrol bindingpocket. D, ribbon view of the Hsp90-celastrol bindingpocket. Only amino acid residues close to celastrol are displayed for clarity. E, ribbon view of the Hsp90-Cdc37-celastrol bindingpocket. Only amino acid residues close to celastrol are displayed for clarity. F, ribbon view of the superimposition of Hsp90-celastrol (brown) complex with the Hsp90-p23/Sba1 (blue) X-ray structure. AMPPNP, ATP analogue; yellow, ‘‘lid’’ segment for HSP90-celastrol; pink, ‘‘lid’’ segment for Hsp90-p23. The p23/Sba1 cochaperone that is also present in the crystal has been omitted for clarity. MolCancerTher2008;7(1).January2008 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. 164 Novel Hsp90 Inhibitor to Disrupt Hsp90/Cdc37 Complex Western Blotting and Immunoprecipitation The procedure for the Western blot analysis was briefly described as follows. After drug treatment, cells were washed twice with ice-cold PBS, collected in lysis buffer [20 mmol/L Tris (pH 7.5), 1% NP40, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L Na3VO4] supplemented with a protease inhibitor mixture (Sigma; added at a 1:100 dilution), and incubated on ice for 20 min. The lysate was then centrifuged at 14,000 Â g for 10 min. Equal amounts of total protein were subjected
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