Cancer Research The Future of Cancer Research: Science and Patient Impact 09 AM Call for Abstracts
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, J.
Right arrow Articles by Sebti, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, J.
Right arrow Articles by Sebti, S. M.
[Cancer Research 61, 1280-1284, February 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

CEP1612, a Dipeptidyl Proteasome Inhibitor, Induces p21WAF1 and p27KIP1 Expression and Apoptosis and Inhibits the Growth of the Human Lung Adenocarcinoma A-549 in Nude Mice1

Jiazhi Sun, Sangkil Nam, Chang-Sun Lee, Benyi Li, Domenico Coppola, Andrew D. Hamilton2, Q. Ping Dou2 and Saïd M. Sebti2,3

Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Departments of Biochemistry and Molecular Biology [J. S., S. N., B. L., Q. P. D., S. M. S.] and Pathology [D. C.], University of South Florida, Tampa, Florida 33612, and Department of Chemistry, Yale University, New Haven, Connecticut 06511 [C-S. L., A. D. H.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
The proteasome is a high molecular weight multicatalytic protease complex. This 20S proteasome (Mr 700,000) associates with activators to form a 26S proteasome (Mr 2,000,000), which catalyzes the ATP-dependent degradation of polyubiquitinated protein substrates (1, 2, 3) . The multicatalytic complex has several proteolytic activities including chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolyzing, small neutral amino acids-preferring, and branched chain amino acid-preferring activities. Among the proteasome substrates are proteins intimately involved in the regulation of several important pathways including those of programmed cell death (apoptosis), cell division cycle, signal transduction, and antigen presentation (reviewed in Refs. 4 –8). Aberrant proteasome activity has been implicated in several pathological conditions such as cancer, muscular dystrophy, emphysema, leprosy, and Alzheimer’s disease (7 , 9) . Of particular interest to us is the involvement of the proteasome in the degradation of proteins pivotal to the cell division cycle and apoptosis in human cancer cells. For example, the degradation by the proteasome of cyclins B and E is required for exit out of mitosis and entry into the S-phase of the cell cycle, respectively. The proteasome is also responsible for the degradation of proteins involved in the regulation of cell survival/apoptosis (i.e., p53 and nuclear factor-{kappa}B) as well as those involved in the interface between cell cycle and apoptosis (i.e., the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, Refs. 4 –8).

The fact that the proteasome has been implicated in many disease states suggested that proteasome inhibitors have therapeutic potential. Furthermore, these inhibitors would provide outstanding tools for determining the role of the proteasome in various physiological functions. This has prompted many to design, synthesize, and biologically evaluate proteasome inhibitors.

Several groups have made a variety of structurally unrelated proteasome inhibitors (6 , 10) . These include peptide aldehydes such as PSI (N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-Leu-H (11) or CbzLLnV-H and CbzLLnL-H (12) , which inhibit the chymotrypsin-like activity of the proteasome. Others have made tripeptide epoxyketones (13) , dipeptidyl boronic acid (14) , indanylamide derivatives (15) , and vinyl sulfone derivatives (16) .

Iqbal et al. (17) designed a series of dipeptidyl aldehydes that inhibited potently the chymotrypsin-like activity of the proteasome. One of these, CEP1612,4 was highly selective (>500-fold) for chymotrypsin-like over trypsin-like activity of the proteasome. CEP1612 was also shown to be cell permeable and inhibited the proteasome in intact cells with an IC50 of 1 µM. Initially, CEP1612 was shown to block MCH-1 antigen processing (18) . Subsequently, An et al. (19) demonstrated that CEP1612 induces apoptosis in human prostate, breast, tongue, and brain cancer cell lines. Furthermore, they demonstrated that CEP1612 induced apoptosis in SV40-transformed but not the parental normal human fibroblasts WI-38 (19) . In addition, inhibition of the proteasome activity is sufficient to overcome Bcl-2- or Bcr-Abl-mediated drug resistance of tumor cells (19 , 20) . In this study, human lung adenocarcinoma A-549 cells were implanted s.c. in nude mice to evaluate whether CEP1612 can induce apoptosis and inhibit human tumor growth in vivo.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Synthesis of Dipeptidyl Proteasome Inhibitor CEP-1612.
CEP1612 was prepared according to the procedure reported previously (17) , except for a key modification in the synthesis of the NH2-terminal region. Benzyl cyclopentylacetate was monoalkylated with 1,8-diiodooctane in tetrahydrofuran by sodium hexamethyldisilazide at -78°C. Substitution with potassium phthalimide and deprotection of the benzyl ester produced the NH2-terminal derivative. This was coupled with a COOH-terminal moiety using an acid chloride activation method. Final deprotection by 1 N HCl produced the aldehyde, CEP1612.

Cell Culture and Drug Treatment.
Human lung adenocarcinoma A-549 cells were purchased from American Type Culture Collection (Rockville, MD) and grown in Ham’s F-12K Nutrient Mix, containing 10% FBS, at 37°C in a humidified incubator containing 10% CO2. Treatment of cells with the proteasome inhibitor CEP1612 was performed as described previously (19) .

Proteasome Activity Assay.
Chymotrypsin-like substrate suc-Leu-Leu-Val-Tyr-AMC and 20S proteasome (recombinant, Methanosarcina thermophila, Escherichia coli) were purchased from Calbiochem (La Jolla, CA). For measurement of 20S proteasome activity in vitro, Suc-Leu-Leu-Val-Tyr-AMC (20 µM), 0.5 µg of 20S proteasome, and inhibitor CEP1612 (0.05–10 µM) were incubated in 100 µl of assay buffer (20 mM HEPES, 0.5 mM EDTA, and 0.035% SDS, pH 8.0) for 1.5 h at 37°C. Reaction samples were diluted to 200 µl with assay buffer. Free AMC liberated by the substrate hydrolysis was quantified on a fluorometer (VersaFluor fluorometer; Bio-Rad, Richmond, CA) with an excitation filter of 380 nm and an emission filter of 460 nm. For estimation of proteasome activity in tumor tissue extracts from nude mice treated and untreated with CEP1612, tumor extracts (30 µg) and 20 µM Suc-Leu-Leu-Val-Tyr-AMC were incubated in 200 µl of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) at specific time intervals at 37°C. Free AMC was quantified as described above.

Western Blotting.
Whole-cell extraction and the enhanced chemiluminescence Western blot assay were performed as we described previously (21) . Briefly, proteins were resolved by 15 or 8% SDS-PAGE gel and immunoblotted with antibodies against caspase-3 (CPP32), p21WAF1 (SX118), p27KIP1 (G173-524; PharMingen, San Diego, CA), and PARP (Boehringer Mannheim). The ECL blotting system (NEN Life Science Products, Boston, MA) was used for detection of positive antibody reactions.

Antitumor Activity in the Nude Mouse Tumor Xenograft Model.
Nude mice (Charles River, Wilmington, MA) were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. A-549 cells were harvested, resuspended in PBS, and injected s.c. into the right and left flank (10 x 106 cells/flank) of 8-week-old female nude mice as reported previously (22) . When tumors reached about 100–150 mm3, animals were dosed i.p. with 0.2 ml once daily. Control animals received a saline vehicle, whereas treated animals received injections of CEP1612 (10 mg/kg/day). The tumor volumes were determined by measuring the length (l) and the width (w) and calculating the volume (V = lw2/2), as described previously (22) . Statistical significance between control and treated groups was evaluated by using Student’s t test (P < 0.05).

Effects of CEP1612 on Organ Proteasome Activity.
The ability of CEP1612 to reach mice organs was determined by injecting nude mice with CEP1612 (10 mg/kg i.p. once) and sacrificing the mice at times 0, 5, 15, 60, and 120 min after injection. Three mice were collected per time point. Livers, kidneys, and lungs were then collected and processed for proteasome activity assay as described above for tumor proteasome activity measurements.

TUNEL Assay and Immunostaining.
Apoptosis was determined by TUNEL using an in situ cell death detection kit (Boehringer Mannheim). Frozen sections were prepared from the treated and untreated tumors. The slides were fixed in paraformaldehyde (4% in PBS, pH 7.4). After rinsing with PBS and incubation in permeabilization solution, the tissues were cross reacted with TUNEL reaction mixture (for 60 min at 37°C in a humidified chamber), with converter-alkaline phosphatase solution (for 30 min at 37°C in a humidified chamber), and with alkaline phosphatase substrate solution (Vector Laboratories, Burlington, MA, for 5–10 min). For the immunostaining of p21WAF1 and p27KIP1, the tumor frozen sections and slides were prepared as described for TUNEL assay; the anti-p21WAF1 or anti-p27KIP1 antibody was applied to the slide. The reactions were analyzed by light microscopy.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
CEP1612, a cell permeable dipeptidyl aldehyde proteasome inhibitor, was initially designed and synthesized by Iqbal et al. (17) and subsequently shown to block MCH-1 antigen processing (18) . More recently, An et al. (19) demonstrated that CEP1612 induced apoptosis in several human cancer cell lines and that normal cells were resistant to CEP1612-induced apoptosis. In this report, we present evidence for the ability of CEP1612 to induce the expression of p21WAF1 and p27KIP1 and apoptosis as well as inhibit tumor growth of the human lung adenocarcinoma A-549 in vivo using the nude mouse xenograft model.

To determine the in vivo apoptotic and antitumor activity of CEP1612, we synthesized this molecule as described previously by Iqbal et al. (17) , using modifications of the route as described in "Materials and Methods," and confirmed its ability to inhibit proteasome activity (Fig. 1)Citation . CEP1612 inhibition of the chymotrypsin-like proteasome activity was demonstrated using a recombinant 20S proteasome and Suc-leu-leu-val-Tyr-AMC as substrate as described in "Materials and Methods." Fig. 1BCitation shows that CEP1612 inhibited the proteasome activity in a concentration-dependent manner with an IC50 of 60 nM. Our IC50 is higher than Iqbal et al. (17) , and this could be because of the fact that they used human brain 20S proteasomes and methoxysuccinyl-Glu-Val-Lys-Met-para-nitroanilide as substrate. More important, however, is the fact that CEP1612 synthesized by us was as potent as that synthesized by Iqbal et al. (17) when used in whole cells against the breast carcinoma MCF-7 and SV40-transformed WI-38 (see below).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. A, structure of the dipeptidyl proteasome inhibitor, CEP1612. B, CEP1612 inhibits the 20S proteasome activity in vitro. To determine whether the dipeptidyl proteasome inhibitor CEP1612 blocks proteasome activity in vitro, a cell-free system was used by incubation of the inhibitor with a recombinant 20S proteasome from E. coli and chymotrypsin-like substrate. The proteasome activity was determined as RFU by measuring with a fluorometer the fluorescence from the AMC cleaved product as described in "Materials and Methods." Data are representative of three independent experiments. Bars, SD.

 
We next investigated whether CEP1612 inhibits proteasome activity and induces apoptosis in cultured A-549 cells. A-549 cells were treated with CEP1612 (30 µM) for 24 h and lysed, and the lysates were immunoblotted with antibodies to p21WAF1 and p27KIP1 as described in "Materials and Methods." p21WAF1 and p27KIP1 are two cyclin-dependent kinase inhibitors that are known natural substrates for the chymotrypsin-like activity of the proteasome (23 , 24) . Fig. 2ACitation shows that treatment with CEP1612 resulted in the accumulation of both p21WAF1 and p27KIP1. These data confirmed that CEP1612 inhibits proteasome activity in A-549 cells. Previously, An et al. (19) reported that CEP1612 induces apoptosis in human breast, prostate, brain, and head and neck cancer cell lines. To determine whether CEP1612 induces programmed cell death in A-549 cells, we exposed these cells to CEP1612 and determined its ability to induce processing and activation of caspase-3 and cleavage of PARP as described in "Materials and Methods." Fig. 2BCitation shows that treatment with CEP1612 resulted in the processing of caspase-3, as evident from the CEP1612-dependent generation of the p17 form from the CPP32 form. Similarly, CEP1612 treatment also resulted in the cleavage of PARP to its p85 proteolytic fragment (Fig. 2B)Citation . The IC50 of CEP1612 to induce PARP cleavage in A-549, MCF-7, and SV40-transformed WI-38 was 1 µM and similar to what An et al. (19) reported previously.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2. The proteasome inhibitor CEP1612 results in accumulation of cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1 and induces apoptosis in vitro. A, accumulation of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1 in A-549 cells treated either with DMSO vehicle or 30 µM CEP1612 for 24 h is shown. Whole-cell extracts were prepared and analyzed by Western blot using anti-p21WAF1 and anti-p27KIP1 antibodies. B, activation of caspase-3 and stimulation of PARP cleavage in A-549 cells treated with 30 µM CEP1612 for 24 h. Immunoblotting was performed using anti-CPP32 and anti-PARP antibodies. The full-length PARP and Mr 85,000 fragment (p85/PARP) as well as pro-CPP32 (CPP32) and activated form (p17/CPP32) are indicated. Data are representative of two independent experiments.

 
The data described above clearly demonstrate that the proteasome inhibitor CEP1612 induces apoptosis in cultured A-549 cells. To determine CEP1612 antitumor efficacy and its ability to induce apoptosis in vivo, we implanted A-549 cells s.c. in nude mice, and when the tumors were palpable (100–150 mm3), the mice were either treated with vehicle control or CEP1612 (10 mg/kg/day, i.p.). After 1 month of daily treatment, the A-549 tumors were removed and processed for inhibition of proteasome activity and TUNEL as well as p21WAF1 and p27KIP1 immunostaining as described in "Materials and Methods." Fig. 3ACitation shows that tumors from control animals grew to an average size of 285.3 ± 26.0 mm3. In contrast, tumors from CEP1612-treated animals grew to an average size of only 176.4 ± 36.6 mm3. Thus, treatment with CEP1612 resulted in a statistically significant (P < 0.05), 68% tumor growth inhibition (Fig. 3A)Citation . To demonstrate that the proteasome activity of A-549 tumor cells was inhibited in vivo, tumor extracts were incubated with purified peptide substrate for various periods of time, and the chymotrypsin-like activity of the proteasome was assayed as described in "Materials and Methods." Fig. 3BCitation shows that tumor extracts from control animals contained proteasome activity that reached 2000 RFUs over 120 min. In contrast, tumor extracts from CEP1612-treated animals contained only 1050 RFUs, demonstrating that CEP1612 treatment resulted in 43% inhibition of proteasome activity in vivo (Fig. 3B)Citation . To further document that CEP1612 inhibited the proteasome activity, we next determined by immunostaining whether treatment of nude mice with this molecule resulted in the accumulation in the A-549 tumors of two of its natural substrates, p21WAF1 and p27KIP1. Fig. 4ACitation shows that tumors from control animals have low levels of p21WAF1 and p27KIP1. However, tumors from CEP1612-treated animals contained much higher levels of these cyclin-dependent kinase inhibitors. Consistent with our in vitro results of Fig. 2BCitation , CEP1612 treatment in vivo was also able to induce accumulation of p21WAF1 and p27KIP1. Thus, CEP1612 injected i.p. was able to reach the s.c.-implanted A-549 tumors and inhibit the intended target in vivo. We next determined whether CEP1612 also inhibited the proteasome activity in mice organs. To this end, we have injected i.p. 15 nude mice with CEP1612 (10 mg/kg) and sacrificed the mice after 5, 15, 60, and 120 min (3 mice/time point). Livers, lungs, and kidneys were then processed for proteasome activity assays as described in "Materials and Methods." CEP1612 inhibited potently (80%) liver proteasome activity within 5 min of treatment. In contrast, kidney proteasome activity was inhibited by only 50% 2 h after CEP1612 injection. Lung proteasome activity was not affected by CEP1612 treatment (Fig. 4C)Citation .



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. A, antitumor efficacy of CEP1612 against human lung adenocarcinoma A-549 xenografts in nude mouse. A-549 cells were injected s.c. at day 0 into the right and left flanks (10 x 106 cells/flank) of 8-week-old female nude mice. When the tumor size reached an average of 125 mm3, animals (five mice/group) were treated i.p. daily either with vehicle (•) or CEP1612 (10 mg/kg; {blacktriangledown}) for 31 days, and tumor volumes were determined as described in "Materials and Methods." Data are representative of two independent experiments. Statistical significance between control and treated groups were evaluated by using Student’s t test (P < 0.05). Bars, SE. B, CEP1612 inhibits SE proteasome enzyme activity in vivo. Nude mice were treated as described in A. At the end of experiment, tumor specimens were dissected, frozen, and sectioned. Then tumor extracts from CEP1612 treated ({blacktriangleup}), untreated ({blacksquare}), or BSA control (•) were incubated with purified chymotrypsin-like substrates at various times (0–120 min), and the proteasome activity was determined using a fluorometer as described in "Materials and Methods." Data are representative of two independent experiments; Bars, SD.

 


View larger version (106K):
[in this window]
[in a new window]
  		Fig. 4. CEP1612 accumulates the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1 and induces apoptosis in vivo. A, A-549 xenografts in nude mice were treated and prepared as described in the Fig. 3Citation legend, and immunostaining was performed by using anti-p21WAF1 and anti-p27KIP1 antibodies. B, to determine whether administration of CEP1612 induces apoptosis of A-549 cells in vivo, nude mice bearing A-549 tumors were treated either with vehicle (DMSO) or CEP1612 (10 mg/kg/day, i.p.) for 31 days and sacrificed, and tumor specimens were dissected, frozen, and sectioned; apoptosis was detected using an in situ TUNEL assay as described in "Materials and Methods." Areas of the tumor that are apoptotic show dark nuclear staining, and p21WAF1 and p27KIP1 also show intense staining. The fields shown are x 100. C, to determine whether CEP1612 inhibits the intense proteasome activity in mice organs, we treated the mice with CEP1612 for various periods of time and collected lungs, livers, and kidneys and measured their proteasome activity as described in "Materials and Methods." Data are representative of two independent experiments; Bars, SD.

 
The above results demonstrated that CEP1612 reaches its target and inhibits human tumor growth in vivo. Whether this inhibition of tumor growth is attributable to induction of apoptosis was next determined. Tumor specimens from control and CEP1612-treated animals were processed for TUNEL assay as described in "Materials and Methods." TUNEL analysis demonstrated that A-549 tumors from vehicle control-treated animals contain no apoptotic cells (Fig. 4B)Citation . In contrast, tumor cells from animals treated with CEP1612 were apoptotic. Thus, CEP1612 treatment resulted in the induction of massive apoptosis in A-549 tumors in vivo.

Taken together, our data demonstrate that treatment of A-549 tumor-bearing nude mice i.p. with CEP1612 resulted in inhibition of the proteasome activity in these tumors, accumulation of cyclin-dependent kinase inhibitors, induction of programmed cell death, and significant inhibition of tumor growth. However, despite induction of apoptosis, no tumor regression was observed, suggesting possibly that greater plasma levels may be required. Alternatively, a subpopulation of the tumor cells may be resistant to the proteasome inhibitor. Therefore, the long-term effect on tumors may be tumor growth delay rather than tumor regression. With regard to side effects, over the 31-day period of daily i.p. treatment, no overall gross toxicity was observed. Indeed, animals treated with CEP1612 (10 mg/Kg/day; 31 days) showed no weight loss, decreased activity, or anorexia. This is consistent with previous studies from An et al. (19) that demonstrated that in cultured cells, human cancer cells were sensitive whereas normal cells were resistant to CEP1612-induced apoptosis. However, our pharmacodynamic studies demonstrated that the proteasome inhibitor was able to reach several organs and inhibit their proteasome activity. Therefore, more detailed microscopic and macroscopic pathology studies are required to document the lack of toxicity of this proteasome inhibitor.

Recently, Orlowski et al. (25) and Adams et al. (26) , using structurally unrelated molecules, also demonstrated the ability of proteasome inhibitors to inhibit tumor growth. Orlowski et al. (25) showed that treatment of Burkitt’s lymphoma-bearing nude mice with Z-LLF-CHO inhibited tumor growth by 42%. Similarly, Adams et al. (26) showed that the dipeptide boronic acid PS-341 inhibited by 60% the growth in nude mice of the prostate cancer cell line PC-3. The fact that three structurally unrelated proteasome inhibitors can inhibit human tumor growth in vivo gives strong support for proof-of-concept of using proteasome inhibitors as novel anticancer drugs. The remaining challenge is to design more potent and selective proteasome inhibitors, with optimal pharmacokinetic profiles to suppress human tumor growth without toxicity in clinical settings.


   ACKNOWLEDGMENTS
 
We thank the Pathology Core facility at the H. Lee Moffitt Cancer Center and Research Institute for processing the immunostaining samples.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by research grants from the National Cancer Insitute (to S. M. S. and A. D. H) and the National Institute of Aging (to Q. P. D.) H. Lee Moffitt Cancer Center and Research Institute (to Q. P. D. and S. M. S.). Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612-9494. Back

4 The abbreviations used are: CEP1612, phthalimide-(CH2)8CH(cyclopentyl) CO-Arg(NO2)-Leu-H, CKI, cyclin-dependent kinase inhibitor, TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; PARP, poly(ADP-ribose) polymerase; RFU, relative fluorescent unit. Back

Received 11/21/00. Accepted 1/ 4/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Coux O., Tanaka K., Goldberg A. L. Structure and functions of the 20S and 26S proteasome.. Annu. Rev. Biochem., 65: 801-847, 1996.[Medline]
  2. Goldberg A. L. Functions of the proteasome: the lysis at the end of the tunnel.. Science (Washington DC), 268: 522-523, 1995.[Free Full Text]
  3. DeMartino G. N., Slaughter C. A. The proteasome, a novel protease regulated by multiple mechanisms.. J. Biol. Chem., 274: 22123-22126, 1999.[Free Full Text]
  4. Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation.. Curr. Opin. Cell Biol., 7: 215-223, 1995.[Medline]
  5. Baumeister W., Walz J., Zuhl F., Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease.. Cell, 92: 367-380, 1998.[Medline]
  6. Lee D. H., Goldberg A. L. Proteasome inhibitors: valuable new tools for cell biologists.. Trends Cell Biol., 8: 397-403, 1998.[Medline]
  7. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life.. EMBO J., 17: 7151-7160, 1998.[Medline]
  8. Dou Q. P., Li B. Proteasome inhibitors as potential novel anticancer agents.. Drug Resistance Updates, 2: 215-223, 1999.[Medline]
  9. Spataro V., Norbury C., Harris A. L. The ubiquitin-proteasome pathway in cancer.. Br. J. Cancer, 77: 448-455, 1998.[Medline]
  10. Terrett N. Inhibition of the proteasome as a therapeutic approach.. Drug Discovery Today, 4: 188-192, 1999.[Medline]
  11. Figueiredo-Pereira M. E., Berg K. A., Wilk S. A. New inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell.. J. Neurochem., 63: 1578-1581, 1994.[Medline]
  12. Rock K. L., Gramm C., Rothstein L., Clark K., Stein R., Dick L., Hwang D., Goldberg A. L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.. Cell, 78: 761-771, 1994.[Medline]
  13. Spaltenstein A., Leban J. J., Huang J. J., Reinhardt K. R., Viveros O. H., Sigafoos J., Crouch R. Design and synthesis of novel protease inhibitors tripeptide {alpha}',ß'-epoxyketones as nanomolar inactivators of the proteasome.. Tetrahedron Lett., 37: 1343-1346, 1996.
  14. Adams J., Behnke M., Chen S., Cruickshank A. A., Dick L. R., Grenier L., Klunder J. M., Ma Y. T., Plamondon L., Stein R. L. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids.. Bioorg. Med. Chem. Lett., 8: 333-338, 1998.[Medline]
  15. Lum R. T., Kerwar S. S., Meyer S. M., Nelson M. G., Schow S. R., Shiffman D., Wick M. M., Joly A. A new structural class of proteasome inhibitors that prevent NF-{kappa} B activation. Biochem. Pharmacol., 55: 1391-1397, 1998.[Medline]
  16. Bogyo M., Shin S., McMaster J. S., Ploegh H. L. Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes.. Chem. Biol., 5: 307-320, 1998.[Medline]
  17. Iqbal M., Chatterjee S., Kauer J. C., Das M., Messina P., Freed B., Biazzo W., Siman R. Potent inhibitors of proteasome.. J. Med. Chem., 38: 2276-2277, 1995.[Medline]
  18. Harding C. V., France J., Song R., Farah J. M., Chatterjee S., Iqbal M., Siman R. Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-I antigen-processing pathway.. J. Immunol., 155: 1767-1775, 1995.[Abstract]
  19. An B., Goldfarb R. H., Siman R., Dou Q. P. Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts.. Cell Death Differ., 5: 1062-1075, 1998.[Medline]
  20. Dou Q. P., McGuire T. F., Peng Y., An B. Proteasome inhibition leads to significant reduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 human chronic myelogenous leukemia cells.. J. Pharmacol. Exp. Ther., 289: 781-790, 1999.[Abstract/Free Full Text]
  21. An B., Dou Q. P. Cleavage of retinoblastoma protein during apoptosis: an interleukin 1ß-converting enzyme-like protease as candidate.. Cancer Res., 56: 438-442, 1996.[Abstract/Free Full Text]
  22. Sun J., Qian Y., Hamilton A. D., Sebti S. M. Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts. Oncogene, 16: 1467-1473, 1998.[Medline]
  23. Blagosklonny M. V., Wu G. S., Omura S., el-Deiry W. S. Proteasome-dependent regulation of p21WAF1/CIP1 expression. Biochem. Biophys. Res. Commun., 227: 564-569, 1996.[Medline]
  24. Pagano M., Tam S. W., Theodoras A. M., Beer-Romero P., Del Sal G., Chau V., Yew P. R., Draetta G. F., Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.. Science (Washington DC), 269: 682-685, 1995.[Abstract/Free Full Text]
  25. Orlowski R. Z., Eswara J. R., Lafond-Walker A., Grever M. R., Orlowski M., Dang C. V. Tumor growth inhibition induced in a murine model of human Burkitt’s lymphoma by a proteasome inhibitor.. Cancer Res., 58: 4342-4348, 1998.[Abstract/Free Full Text]
  26. Adams J., Palombella V. J., Sausville E. A., Johnson J., Destree A., Lazarus D. D., Maas J., Pien C. S., Prakash S., Elliott P. J. Proteasome inhibitors: a novel class of potent and effective antitumor agents.. Cancer Res., 59: 2615-2622, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
V. Milacic, S. Banerjee, K. R. Landis-Piwowar, F. H. Sarkar, A. P.N. Majumdar, and Q. P. Dou
Curcumin Inhibits the Proteasome Activity in Human Colon Cancer Cells In vitro and In vivo
Cancer Res., September 15, 2008; 68(18): 7283 - 7292.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
B. Li, A. Sun, H. Youn, Y. Hong, P. F. Terranova, J.B. Thrasher, P. Xu, and D. Spencer
Conditional Akt activation promotes androgen-independent progression of prostate cancer
Carcinogenesis, March 1, 2007; 28(3): 572 - 583.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
V. Milacic, D. Chen, L. Ronconi, K. R. Landis-Piwowar, D. Fregona, and Q. P. Dou
A Novel Anticancer Gold(III) Dithiocarbamate Compound Inhibits the Activity of a Purified 20S Proteasome and 26S Proteasome in Human Breast Cancer Cell Cultures and Xenografts
Cancer Res., November 1, 2006; 66(21): 10478 - 10486.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Yang, D. Chen, Q. C. Cui, X. Yuan, and Q. P. Dou
Celastrol, a Triterpene Extracted from the Chinese "Thunder of God Vine," Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice.
Cancer Res., May 1, 2006; 66(9): 4758 - 4765.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Bazzaro, M. K. Lee, A. Zoso, W. L.H. Stirling, A. Santillan, I.-M. Shih, and R. B.S. Roden
Ubiquitin-proteasome system stress sensitizes ovarian cancer to proteasome inhibitor-induced apoptosis.
Cancer Res., April 1, 2006; 66(7): 3754 - 3763.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
Y.-W. Chen, S.-C. Huang, S.-Y. Lin-Shiau, and J.-K. Lin
Bowman-Birk inhibitor abates proteasome function and suppresses the proliferation of MCF7 breast cancer cells through accumulation of MAP kinase phosphatase-1
Carcinogenesis, July 1, 2005; 26(7): 1296 - 1306.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. A. Williams and D. J. McConkey
The Proteasome Inhibitor Bortezomib Stabilizes a Novel Active Form of p53 in Human LNCaP-Pro5 Prostate Cancer Cells
Cancer Res., November 1, 2003; 63(21): 7338 - 7344.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. Hideshima, C. Mitsiades, M. Akiyama, T. Hayashi, D. Chauhan, P. Richardson, R. Schlossman, K. Podar, N. C. Munshi, N. Mitsiades, et al.
Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341
Blood, February 15, 2003; 101(4): 1530 - 1534.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Gaedicke, E. Firat-Geier, O. Constantiniu, M. Lucchiari-Hartz, M. Freudenberg, C. Galanos, and G. Niedermann
Antitumor Effect of the Human Immunodeficiency Virus Protease Inhibitor Ritonavir: Induction of Tumor-Cell Apoptosis Associated with Perturbation of Proteasomal Proteolysis
Cancer Res., December 1, 2002; 62(23): 6901 - 6908.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
Y.-C. Huang, L.-Y. Chuang, and W.-C. Hung
Mechanisms Underlying Nonsteroidal Anti-Inflammatory Drug-Induced p27Kip1 Expression
Mol. Pharmacol., December 1, 2002; 62(6): 1515 - 1521.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Z. Orlowski, G. W. Small, and Y. Y. Shi
Evidence That Inhibition of p44/42 Mitogen-activated Protein Kinase Signaling Is a Factor in Proteasome Inhibitor-mediated Apoptosis
J. Biol. Chem., July 26, 2002; 277(31): 27864 - 27871.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, J.
Right arrow Articles by Sebti, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, J.
Right arrow Articles by Sebti, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online