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NPI-0052 Enhances Tumoricidal Response to Conventional Cancer Therapy in a Colon Cancer Model

Authors' Affiliations: ¹ Division of Surgical Oncology, Massachusetts General Hospital; ² Harvard Medical School, Boston, Massachusetts; and ³ Nereus Pharmaceuticals, San Diego, California

Requests for reprints: James C. Cusack, Jr., Division of Surgical Oncology, Massachusetts General Hospital, Yawkey 7th Floor, 55 Fruit Street, Boston, MA 02114. Phone: 617-724-4093; Fax: 617-724-3895; E-mail: jcusack{at}partners.org.

	Abstract

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Purpose: In the current study, we examine the effects of a novel proteasome inhibitor, NPI-0052 (salinosporamide A), on proteasome function and nuclear factor-B activation and evaluate its ability to enhance treatment response in colon cancer xenografts when administered orally.

Experimental Design: The effects of treatment on nuclear factor-B activation, cell cycle regulation, and apoptosis were determined. The pharmacodynamic effect of NPI-0052 on 20S proteasome function was assayed in vivo following oral and i.v. drug administration and compared with treatment with bortezomib. The effect of combined treatment with chemotherapy was determined in a colon cancer xenograft model.

Results: We found that NPI-0052 is a potent, well-tolerated proteasome inhibitor that has pharmacodynamic properties distinct from bortezomib in that it achieves significantly higher and more sustained levels of proteasome inhibition. When combined with chemotherapy, NPI-0052 increases apoptosis and shifts cells toward G₂ cell cycle arrest. When added to chemotherapy in vivo [using combinations of 5-fluorouracil (5-FU), CPT-11, Avastin (bevacizumab), leucovorin, and oxaliplatin], NPI-0052 significantly improved the tumoricidal response and resulted in a 1.8-fold increased response to CPT-11, 5-FU, and leucovorin triple-drug combination (P = 0.0002, t test), a 1.5-fold increased response to the oxaliplatin, 5-FU, and leucovorin triple-drug combination (P = 0.013, t test), and a 2.3-fold greater response to the CPT-11, 5-FU, leucovorin, and Avastin regimen (P = 0.00057).

Conclusions: The high level of proteasome inhibition achieved by NPI-0052 is well tolerated and significantly improves the tumoricidal response to multidrug treatment in a colon cancer xenograft model. Further evaluation of this novel proteasome inhibitor in clinical trials is indicated.

	Materials and Methods

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Cell culture, plasmids, and transfections. The human colorectal cancer cell line LoVo and the breast cancer cell line BT-474 were obtained from American Type Culture Collection (Rockville, MD) and grown according to American Type Culture Collection instructions. Cell cultures were maintained in 5% CO₂ at 37°C. HeLa cells were grown in Eagle's MEM with 2 mmol/L L-glutamine and Earle's balanced salt solution adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential amino acids, 1.0 mmol/L sodium pyruvate, and 10% heat-inactivated horse serum at 37°C and 5% CO₂. The NF-B/Luc cells were prepared by selecting transfected HeLa cells that carry a luciferase reporter gene under the regulation of 5x NF-B binding sites.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay was done to evaluate NF-B activation and inhibition. Nuclear protein was extracted from LoVo colorectal cancer cells at 2 and 4 hours after the cells were treated with or without prior exposure to NPI-0052 (200 nmol/L, Nereus Pharmaceuticals, Inc., San Diego, CA). The DNA probe used contains a NF-B binding site (5'-GGGATTCCC-3'). Six milligrams of nuclear extracts were separated on a 5% polyacrylamide gel with preincubation of 1 mg of poly(deoxyinosinic-deoxycytidylic acid) and 20,000 cpm of ³²P-labeled DNA probe in a binding buffer at room temperature for 15 minutes. Positive control was BT-474 cells treated with 10 µg/mL doxorubicin (Sigma Co., St. Louis, MO) for 2 hours.

Drug treatment of HEK 293 reporter cells and lysate preparation. HEK 293 NF-B/Luc cells were treated with 50 or 500 nmol/L of NPI-0052 for 1 hour and then stimulated with 10 ng/mL of recombinant human tumor necrosis factor- (TNF-; US Biological, Swampscott, MA) for 30 minutes. DMSO was used as a control. Cells were then washed once with ice-cold 1x Ca²⁺/Mg²⁺–free Dulbecco's PBS and lysed on ice in radioimmunoprecipitation assay buffer containing 0.9% NaCl, 50 mmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 1 mmol/L EDTA, 0.25% Na-deoxycholic acid, 2 mmol/L Na₃VO₄, and 1x protease inhibitor cocktail (Calbiochem, San Diego, CA). Cell lysates were cleared by centrifugation at 14,000 rpm for 10 minutes, 4°C. Protein concentration of cell lysates was determined with bicinchoninic acid kit (Pierce Biotechnology, Inc., Rockford, IL) before SDS-PAGE loading.

Western blotting of HEK 293 reporter cells and detection of NF-B-dependent luciferase activity in HeLa cells. Equal protein concentrations of cell lysates were resolved on 10% NuPage MES precast gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes according to the instructions of the manufacturer. Membranes were blocked in 5% nonfat dry milk in 1x Dulbecco's PBS containing 0.1% Tween 20 at room temperature for 2 hour. Western blot analyses were done with primary antibodies against IB, phospho-IB (Cell Signaling Technology, Beverly, MA), and tubulin (Lab Vision, Fremont, CA). Antibodies against P21, P27, and P53 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a concentration of 1:500. A horseradish peroxidase–conjugated sheep anti-mouse antibody (Amersham Biosciences, Piscataway, NJ) was used as the secondary antibody and horseradish peroxidase activity was visualized by enhanced chemiluminescence detection system (Pierce Biotechnology). The NF-B-luciferase reporter construct, pNFB-Luc, was purchased from BD Biosciences (Palo Alto, CA). HeLa cells were transiently transfected with pNFB-Luc using FuGENE 6 (Roche, Indianapolis, IN). Twenty-four hours posttransfection, NF-B/luciferase HeLa cells were pretreated with varying concentrations of NPI-0052 or bortezomib for 1 hour and then treated with 10 ng/mL of TNF- (Calbiochem/EMD Biosciences, San Diego, CA) for 6 hours. Four hours later, luciferase activity was measured using the luciferase reporter assay kit from BD Biosciences as previously described (6).

Cell death ELISA. The Cell Death Detection ELISA^plus assay (Roche Applied Sciences) was used to evaluate the effect of proteasome inhibition using NPI-0052 on the apoptotic response to combination therapies. Three different drug combinations were examined including (a) SN-38, the active metabolite of CPT-11 (1 mg/mL; Pfizer, Inc., Groton, CT), 5-fluorouracil (5-FU; 10 mg/mL; America Pharmaceutical Partners, Inc., Schaulburg, IL), and leucovorin (1 mmol/L; Bedford Laboratories, Bedford, OH); (b) oxaliplatin (25 mmol/L; Sanofi-Synthelabo, Inc., New York, NY), 5-FU (10 mg/mL), leucovorin (1 mmol/L); and (c) SN-38 (1 mg/mL), 5-FU (10 mg/mL), leucovorin (1 mmol/L), and Avastin (10 µg/mL; Genentech, Inc., South San Francisco, CA). Each drug was sequentially added to the culture medium 3 hours after the prior drug treatment. When used, NPI-0052 (200 nmol/L) was the last drug added. Whole-cell lysates were obtained from harvested cells 48 hours after exposure to the last drug treatment. The assay was done according to the instructions of the manufacturer and the results were measured at 405 nm with a reference wavelength of 490 nm on a spectrophotometer. The experiment was done in triplicate and apoptosis enrichment was determined as fold change compared with the control group.

Cell cycle analysis by flow cytometry. LoVo cells were synchronized by serum starvation for 72 hours, then treated with SN38 (active metabolite of CPT-11) at a concentration of 1 mg/mL without or with pretreatment (or posttreatment) of NPI-0052 at a concentration of 200 nmol/L. Forty-eight hours after treatment with SN38, cells were collected and fixed with 80% ethanol overnight. Cells were then resuspended in 1 mL of 1% bovine serum albumin in PBS, and 25 mL of 40x propidium iodide (500 mg/mL; Sigma) and 25 mL of RNase A (10 mg/mL) were added for 30 minutes. Analysis was done using a FACScan flow cytometer (BD Biosciences, San Jose, CA). Area under the curve was calculated using ModFit LT version 3.1 software (Varity Software, Topsham, ME).

In vivo proteasome activity assay. NPI-0052 was dissolved in 100% DMSO and serially diluted with 5% Solutol (Solutol HS 15; polyethylene glycol 660 12-hydroxystearate; BASF, Shreveport, LA), yielding a final concentration of 2% DMSO. Vehicle control consisted of 2% DMSO and 98% (5% Solutol). Female C57Bl/6 mice (n = 5) were treated i.v. and male Swiss-Webster mice (n = 5) were treated orally with various concentrations of NPI-0052 at a volume of 10 mL/kg. Bortezomib (1 mg/kg) was administered i.v. also at a dose volume of 10 mL/kg. Twenty microliters of blood were collected via the tail vein after creating a small puncture using a 25-gauge needle. Blood was immediately lysed with 0.5 mmol/L EDTA for 30 minutes on ice and centrifuged at 6,600 x g for 10 minutes at 4°C. The lysates were then removed and analyzed by a fluorescent 20S proteasome assay as previously described (8). Protein content of the samples was determined by a Coomassie protein assay (Pierce). Data are represented as percent inhibition compared with vehicle control treated animals.

In vivo evaluation of tumor growth. Tumor growth was assessed in a LoVo xenograft model. The tumors were established by injecting 5 million cells into the flank of 6-week-old female nu/nu mice. Treatment was initiated once the tumors reached a mean diameter of 8 to 10 mm. CPT-11 (33 mg/kg) was administered i.v. via tail-vein injection twice weekly whereas 5-FU (33 mg/kg), leucovorin (90 mg/kg), and Avastin (2.5 mg/kg) were injected i.p. twice weekly. Oxaliplatin (10 mg/kg i.p.) was injected once during the treatment course and NPI-0052 (0.25 mg/kg) was delivered by oral gavage twice weekly. The maximum tolerated dose of NPI-0052 in mice was 0.50 mg/kg (oral) and 0.25 mg/kg (i.v.). Vehicle was used for the corresponding control group. Tumor size was measured every 4 days and calculated using the formula TV = 4/3r³, where r = 1/2 (mean tumor diameter measured in two dimensions). All experiments were done in full compliance with institutional guidelines and with the approval of the Massachusetts General Hospital Institutional Animal Care and Use Committee.

	Results

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Effects of NPI-0052 treatment on TNF-- and chemotherapy-induced NF-B activation. TNF- and some chemotherapy agents are known to induce the transcription factor NF-B, resulting in the expression of a variety of genes known to be involved in the inhibition of apoptosis (9). To investigate the effects of NPI-0052 treatment on the activation of NF-B, we exposed LoVo cells to NPI-0052 either before or after treatment with SN-38 (Fig. 1A ). The electrophoretic mobility shift assay showed that at both 2 and 4 hours, chemotherapy-induced activation of NF-B was almost completely inhibited by NPI-0052. Following exposure to TNF- and chemotherapy, the inhibitor of NF-B, called IB, is typically phosphorylated, ubiquitinated, and then targeted for degradation by the proteasome. To determine if the inhibition of NF-B was due to changes in its inhibitor IB, NF-B/luciferase HEK 293 cells were exposed to combinations of TNF- and NPI-0052 (Fig. 1B). In these experiments, IB decreased and phospho-IB increased with increasing concentrations of TNF-. The addition of NPI-0052 resulted in an accumulation of phospho-IB rather than its degradation by the proteasome. Furthermore, at the dosage range tested, NPI-0052 was 2-fold more potent inhibitor of TNF--induced NF-B activation compared with bortezomib as measured by the NF-B luciferase reporter assay in HeLa cells (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   Fig. 1. The effects of NPI-0052 on chemotherapy- and TNF-induced NF-B activation were determined. A, electrophoretic mobility shift assay was done to evaluate the effects of treatment on chemotherapy-induced NF-B activation. Bands for the homodimer p50/p50 and p65/p50 heterodimer (classic NF-B) are shown. TNF was used as a positive control. B, Western blot analysis of IB phosphorylation and degradation in HEK 293 cells transfected with the NF-B luciferase reporter plasmid. Lane 4, phosphorylation of IB occurs in response to TNF and accumulates in a dose-dependent manner in the presence of proteasome inhibition using NPI-0052 (lanes 5 and 6). C, inhibition of TNF-induced NF-B-dependent luciferase expression by NPI-0052 was evaluated using a NF-B reporter assay that expresses the firefly luciferase reporter gene (luc) in HeLa cells. Cells were treated with increasing molar concentrations of NPI-0052 or bortezomib for 1 hour, followed by treatment with 10 ng/mL of TNF-. The experiment was done in duplicate and a representative response curve is presented. The IC50 for NPI-0052 was determined to be 36 µmol/L and for bortezomib 100 µmol/L.

View larger version (28K): [in this window] [in a new window]   Fig. 2. A, the cytotoxic effects of combining NPI-0052 to treatment regimens that included various combinations of the chemotherapy agents SN-38 (active metabolite of CPT-11), oxaliplatin (oxali), 5-FU, and leucovorin (Leuk), or the targeted therapy Avastin were evaluated in LoVo cells. The apoptotic response to combination chemotherapy and molecular therapy was detected in vitro by the cell death ELISA assay. The addition of NPI-0052 to all three of the treatment regimens resulted in a significant increase in the apoptotic response. The most dramatic increase in treatment response was observed in the cells treated with oxaliplatin, 5-FU, and leucovorin. B, the cell cycle response to NPI-0052 treatment in SN-38-treated LoVo cells was determined by flow cytometry 48 hours following treatment with SN-38 and NPI-0052. Combined treatment with SN-38 and NPI-0052 resulted in both an increase in the apoptotic cell fraction and a shift toward G2 arrest. C, Western blot analysis was done to evaluate the effects of proteasome inhibition on the expression of cell cycle regulators p21, p27, and p53 in SN-38-treated LoVo cells. Proteasome inhibition using either bortezomib or NPI-0052 resulted in a stabilization of all three cell cycle regulators.

Effects of NPI-0052 on proteasome function in vivo. To evaluate the pharmacodynamics of drug treatment and determine if the level of proteasome inhibition was dose dependent, increasing concentrations of NPI-0052 were administered to mice i.v. and 20S proteasome function was assayed at 1.5 and 24 hours (Fig. 3 ). The degree of proteasome inhibition achieved increased with increasing concentrations of the drug, with a peak level of proteasome inhibition (90%) measured at a drug concentration of 0.2 mg/kg. In comparison, 55% of proteasome inhibition was achieved after bortezomib was administered at the maximum tolerated dose. Furthermore, bortezomib treatment resulted in a higher level of proteasome inhibition at the earlier 1.5 hour time point. In contrast, proteasome inhibition following NPI-0052 treatment gradually increased over the sampled time period and was actually higher at the 24 hour time point for each dosage tested.

View larger version (27K): [in this window] [in a new window]   Fig. 3. The dose and time response of 20S proteasome inhibition in mice was determined at 1.5 and 24 hours after i.v. administration of NPI-0052 (lanes 3-10) or bortezomib (lanes 11 and 12). Columns, mean; bars, SE.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The effect of proteasome inhibition over time was assessed in mice treated with either NPI-0052 (A and B) or bortezomib (C). The inhibition of the 20S chymotrypsin-like proteasome in mice was determined at 1.5, 24, 48, 72, and 168 hours following oral administration of NPI-0052 or i.v. administration of bortezomib. *, P < 0.05, compared with 72 hours; **, P < 0.05, compared with 90 minutes.

View larger version (15K): [in this window] [in a new window]   Fig. 5. A and B, the effect of orally administered NPI-0052 on the inhibition of the 20S proteasome activity in whole blood cells was determined. Mice (n = 5) were administered NPI-0052 orally at 0.25 mg/kg (A) or 0.5 mg/kg (B) on the days indicated. Twenty-four hours after the last treatment, blood was collected, whole blood cell lysates were prepared, and the chymotrypsin-like activity of the 20S proteasome was determined using the peptide substrate suc-LLVY-AMC. The average percent change in body weights at the time of sacrifice is indicated. Body weights were measured to determine potential toxic effects of treatment on animals.

View larger version (20K): [in this window] [in a new window]   Fig. 6. The effect of adding NPI-0052 treatment to three conventional colon cancer therapy regimens was assessed in a colon cancer (LoVo) xenograft model (n = 6 mice per treatment group). Points, mean tumor diameter measured in two dimensions; bars, SE.

	Discussion

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It has previously been shown that inhibitors of NF-B (including proteasome inhibitors), when combined with conventional therapies, augment chemosensitivity by suppressing the NF-B-mediated antiapoptotic response (1–3, 10, 11). We hypothesize that higher levels of proteasome inhibition will result in a higher degree of NF-B inhibition and therefore a greater treatment response. Interestingly, both preclinical and clinical studies using bortezomib have shown that the level of proteasome inhibition achieved at the maximum tolerated dose ranges between 75% and 80%.⁴ Following bortezomib administration in vivo in preclinical studies, lethal toxicity ensues when the level of inhibition of proteasome function exceeded 80% (data not shown). In the current study, in vivo analysis determined that NPI-0052 was significantly more potent than bortezomib in inhibiting 20S proteasome activity. Somewhat surprisingly, we also found that higher levels of proteasome inhibition (approaching 100%) were achieved using NPI-0052, without apparent toxicity.

We also found that NPI-0052 effectively suppressed chemotherapy-induced NF-B activation and augmented apoptosis in vitro, suggesting that the combination of NPI-0052 with conventional chemotherapy may enhance treatment response by muting the NF-B-dependent cancer cell survival signals (3). The cell death ELISA assays and flow cytometric analysis support this hypothesis by showing that the addition of NPI-0052 to chemotherapy in vitro enhanced apoptosis and led to a moderate shift in the cell population toward G₂ arrest. Combined, these data show that higher levels of proteasome inhibition may be achieved with NPI-0052 without inducing nonspecific cytotoxicity. These findings further suggest that this novel compound may be both better tolerated and potentially more effective when used in vivo compared with bortezomib.

Our animal studies show that i.v. and orally administered NPI-0052 is a potent inhibitor of proteasome in vivo, which has unique pharmacodynamic properties compared with bortezomib. We observed a dose- and time-dependent increase in 20S proteasome inhibition in NPI-0052-treated mice. A single dose of NPI-0052 resulted in as high as 90% inhibition of the proteasome compared with 70% inhibition achieved after bortezomib administered at the maximum tolerated dose. NPI-0052 treatment resulted in proteasome inhibition that progressively increased over 24 hours whereas bortezomib reached a maximum level of proteasome inhibition at the 1.5 hour time point and then significantly decreased over the subsequent 24 hours. Further examination of the response over time showed in the case of NPI-0052 that proteasome inhibition after a single dose peaked at 24 hours and remained essentially unchanged for 72 hours. In the case of bortezomib, the proteasome recovers to baseline at 72 hours following bortezomib treatment. The prolonged treatment response seen with NPI-0052 seems to be due to the irreversible binding of the drug to the proteasome compared with reversible binding with bortezomib (1, 2, 5, 7, 12). Importantly, repeated oral dosing of NPI-0052 resulted in 99% inhibition of proteasome function. Surprisingly, this extremely high level of proteasome inhibition was well tolerated and resulted only in a modest amount of weight loss in the animals. This runs counter to our experience with bortezomib where proteasome inhibition exceeding 80% was uniformly lethal in animals within 24 hours (data not shown).

The final and perhaps most important finding from these studies is the ability of NPI-0052 to augment the response to multidrug combination chemotherapy regimens without significantly increasing the toxicity of these complex treatment regimens. Whereas the addition of oxaliplatin, CPT-11, and Avastin to 5-FU-based treatment has contributed to improved survival in patients with metastatic colorectal cancer, the vast majority of patients will ultimately manifest treatment resistance (13, 14). The development of new drug combinations that address chemotherapy resistance is essential to further improvement in survival in metastatic colorectal cancer (13–17). Owing to the multiple systems affected by proteasome function, proteasome inhibition is a logical adjuvant to chemotherapy treatment. Proteasome inhibition not only enhances chemosensitivity via the inhibition of chemotherapy-induced NF-B activation (3, 18) but also decreases vascular endothelial growth factor expression and tumor microvessel density (19) and arrests cancer cell proliferation through effects on cell cycle regulators p21 and p27 (20). Our results show that NPI-0052 effectively augments the therapeutic response to the most commonly used colon cancer multidrug treatment regimens currently used in the clinic. In addition, our study suggests that NPI-0052 may be a superior choice for proteasome inhibition in this treatment paradigm. In summary, our data show that (a) the novel proteasome inhibitor NPI-0052 inhibits proteasome activity in vitro and in vivo; (b) NPI-0052 is orally bioactive; (c) NPI-0052 has a different pharmacodynamic profile compared with bortezomib; (d) NPI-0052 suppresses chemotherapy-induced NF-B activation and overcomes chemotherapy resistance; and (e) high levels of proteasome inhibition induced by NPI-0052 are achievable in vivo and well tolerated. Together, these findings provide the rationale for clinical trials to evaluate the ability of NPI-0052 to enhance treatment efficacy and overcome drug resistance in combination chemotherapy regimens in patients with metastatic colon cancer.

	Footnotes

 
Grant support: National Cancer Institute grants CA77278 and CA98871 (J.C. Cusack, Jr.).

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.

⁴ Unpublished data.

Received 5/12/06; revised 7/27/06; accepted 8/18/06.

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