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1 Division of Hematology/Oncology, Cedars-Sinai Medical Center and UCLA School of Medicine, Los Angeles, California; 2 Department of Pathology, Center for Health Science, UCLA School of Medicine, Los Angeles, California; and Departments of 3 Internal Medicine and 4 Tumor Pathology, KMS, Kochi University, Kochi, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NFB) in DU145 cells, which is a contributor to drug resistance in cancer cells. Furthermore, combination treatment of docetaxel and ritonavir dramatically inhibited the growth of DU145 cells present as tumor xenografts in BNX nude mice compared with either drug alone. Importantly, docetaxel induced expression of CYP3A4 in DU145 xenografts, and ritonavir completely blocked this induction. Ritonavir also inhibited NFB DNA binding activity in DU145 xenografts. Extensive histologic analyses of the liver, spleen, kidneys, bone marrow, skin, and subcutaneous fat pads from these mice showed no abnormalities. In summary, combination therapy of ritonavir and anticancer drugs holds promise for the treatment of individuals with advanced, drug resistant cancers. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of individuals with AIPC (1, 2, 3)
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HIV-1 protease inhibitors (PIs) have become important tools in the management of HIV infection; these include saquinavir mesylate, ritonavir, and indinavir sulfate. Recent studies showed that PIs possess antitumor activity, which is independent from their ability to inhibit HIV protease. We previously found that saquinavir, ritonavir, and indinavir induced growth arrest and differentiation of NB4 and HL-60 human myelocytic leukemia cells and enhanced the ability of all–trans-retinoic acid (ATRA) to decrease proliferation and increase differentiation of these cells (4)
. Other investigators have shown that PIs can decrease proliferation of Kaposi’s sarcoma and prostate cancer cells via inhibition of nuclear factor B (NFB) activity (5, 6, 7)
. We also have recently found that PIs induced growth arrest and apoptosis of multiple myeloma cells via inhibition of signal transducers and activators of transcription 3 and extracellular signal-regulated kinase 1/2 signaling (8)
.
PIs are metabolized by cytochrome P450 3A4 (CYP3A4) in liver microsomes (9) . Interestingly, among the PIs, only ritonavir showed strong CYP3A4 inhibitory effects. Noting this activity, investigators have coadministered ritonavir with saquinavir and found markedly elevated and sustained plasma levels of saquinavir in rat and dog models. This occurred, supposedly, by inhibiting metabolism of saquinavir (10) , and this combination is clinically used for individuals with HIV infection (11) . On the basis of the pharmacokinetics of ritonavir, a coformulated agent containing lopinavir and ritonavir has been developed: Low doses of ritonavir enhanced the activity of lopinavir (12) , and this formulation is being used for first-line therapy for some HIV-infected individuals. Collectively, we hypothesized that ritonavir might enhance the antitumor activity of docetaxel, the latter being a substrate for CYP3A4 (13) . In this study, we found that ritonavir, saquinavir, and indinavir inhibited the growth of the DU145 and PC-3 AIPC cells as measured by the clonogenic assay. Ritonavir blocked the docetaxel-induced expression of CYP3A4at the mRNA level in DU145 cells and enhanced the antitumor effect of docetaxel in vitro and in BNX nude mice bearing DU145 tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Saquinavir mesylate (Roche, Basel, Switzerland), ritonavir (Abbott Labs, North Chicago, IL), and indinavir sulfate (Merck, West Point, PA) were dissolved in dimethyl sulfoxide (Burdick & Jackson, Muskegon, MI) to a stock concentration of 10–2 mol/L and stored at –80°C. Docetaxel (Aventis Pharmaceuticals Inc., Tokyo, Japan) was dissolved in PBS to a stock concentration of 10–4 mol/L and stored at 4°C.
Colony-Forming Assay.
DU145 and PC-3 cells were cultured in a two-layer soft agar system for 14 days as described previously (4)
. Washed single-cell suspensions of cells were enumerated and plated into 24-well flat-bottomed plates with a total of 500 cells/well in a volume of 400 µL/well. The feeder layer was prepared with agar that had been equilibrated at 42°C. Before this step, PIs were pipetted into the wells. After incubation for 14 days, colonies were counted. All of the experiments were done three times using triplicate plates per experimental point.
RNA Isolation and Reverse Transcription-PCR.
Total RNA was isolated as described previously using TRIzol (Life Technologies, Inc.; ref. 4
). One microgram of DNase I-treated RNA was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and 50 ng of the resulting complementary DNAs (cDNAs) were used as templates for PCR. Real-time PCR was carried out by using TaqDNA polymerase (Qiagen, Valencia, CA), 50 ng cDNA for CYP3A4 (500–5 ng in serial dilutions for standard curves), or 1 pg for 18S (10–0.1 pg for standard curve), and SYBR Green I nucleic acid gel staining solution in a 1:60,000 dilution. Primers used for CYP3A4 were 5'-CCTGAGAAGTTCCTCCCTGA-3' and 5'-AATGCAGTTTCTGGGTCCAC-3, which yielded a 99-bp product. PCR conditions were as follows: a 95°C initial activation for 15 minutes followed by 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds, and fluorescence determination at the melting temperature of the product for 20 seconds on an ICycler detection system (Bio-Rad, Hercules, CA). We measured expression of 18S for normalization.
Western Blot Analysis.
DU145 cells (105/mL) were incubated with a variety of concentrations of docetaxel (10–9 to 10–6 mol/L) and ritonavir (10–5 mol/L) either alone or in combination for 24 hours in six-well plates. Lysates were made by standard methods as described previously (14)
. Protein concentrations were quantitated using a Bio-Rad assay. Proteins were resolved on a 4% to 15% SDS polyacrylamide gel, transferred to an Immobilon polyvinylidene difluoride membrane (Amersham Corp., Piscataway, NJ), and probed sequentially with antibodies. Anti-poly(ADP-ribose) polymerase (PARP; Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (Santa Cruz Biotechnology), and ß-actin (Santa Cruz Biotechnology) antibodies were used. The band intensities were measured using densitometry.
Transient Transfection of NFB Small Interfering RNA.
DU145 cells were transiently transfected with NFB small interfering RNA (siRNA; final concentration of 100 nmol/L) using Signalsilence siRNA kit (Cell Signaling Technology, Beverly, MA) according to manufacturer’s instruction. Following transfection, DU145 cells were subjected to Western blot analysis and MTT assay in the presence or absence of docetaxel.
Evaluation of NFB activity by ELISA.
The DNA binding activity of NFB in prostate cancer cells was quantified by ELISA using the trans-AM NFB p65 Transcription Factor Assay kit (Active Motif North America, Carlsbad, CA), according to the instructions of the manufacturer. Briefly, nuclear extracts were prepared as described previously and incubated in 96-well plates coated with immobilized oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') containing a consensus (5'-GGGACTTTCC-3') binding site for the p65 subunit of NFB. NFB binding to the target oligonucleotide was detected by incubation with primary antibody specific for the activated form of p65 (Active Motif North America), visualized by anti-IgG horseradish peroxidase conjugate and developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. Background binding was subtracted from the value obtained for binding to the consensus DNA sequence.
Mice.
Twenty male triple immunodeficient BNX nu/nu mice at 8 weeks of age were purchased from Harlan Sprague Dawley Inc. (Indianapolis, NJ) and were maintained in pathogen-free conditions with irradiated chow.
Treatment Protocol.
Animals were bilaterally, subcutaneously injected with 2 x 106 DU145 cells/tumor in 0.1 mL Matrigel (Collaborative Biomedical Products, Bedford, MA). Mice were divided randomly into four groups of five mice each: group A, control diluent (control); group B, ritonavir; group C, docetaxel; and group D, docetaxel + ritonavir. Ritonavir (10 mg/kg/mouse) was administered five times a week orally. Docetaxel (25 mg/kg/mouse) was administered intravenously once a week. The dose of ritonavir and docetaxel was determined by our preliminary studies. Tumors were measured every week with vernier calipers. Tumor size was calculated by the formula: a x b x c, where "a" is the length and "b" is the width and "c" is the height in millimeters. At the end of the experiment, animals were sacrificed by CO2 asphyxiation, and tumor weights were measured after their careful resection. Blood also was collected from the orbital sinus for chemistry and hematopoietic analysis.
Histology.
Tumors were fixed for 12 hours in 10% neutral buffered formaldehyde after sacrifice; tissue blocks were embedded in paraffin; and H&E-stained sections were examined by light microscopy.
Statistical Analysis.
The statistical significance of the differences was analyzed using the nonparametric Mann-Whitney U test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The most effective PI was ritonavir, which caused a 50% decrease clonal growth of DU145 cells at 3 x 10–6 mol/L (Fig. 1A)
. Ritonavir and saquinavir showed nearly equivalent potency against PC-3 cells with an ED50 of 8 x 10–6 mol/L (Fig. 1B)
. Conversely, none of PIs inhibited growth of normal myeloid committed stem cells (CFU-GM) in soft agar containing cytokines including interleukin 3 and granulocyte macrophage-colony stimulating factor even at a high concentration of PI (2 x 10–5 mol/L; data not shown).
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. Ritonavir (10–5 mol/L) potentiated the effects of docetaxel to activate caspase-3 and cleave its target molecule PARP (Fig. 2B)
. For example, the activated form of caspase-3 and cleaved PARP were negligible in DU145 cells treated with docetaxel (10–9 mol/L) alone; however, the combination of docetaxel (10–9 mol/L) and ritonavir (10–5 mol/L) induced activation of caspase-3 and cleavage of PARP in DU145 cells.
Effect of Ritonavir on Docetaxel-Induced Expression of CYP3A4 Transcripts.
The level of CYP3A4transcripts in DU145 cells was measured using real-time PCR. DU145 cells constitutively expressed CYP3A4, and docetaxel (10–9 mol/L, 24 hours) increased its expression by 2.5-fold (Fig. 3)
. Ritonavir (10–5 mol/L, 24 hours) completely blocked this induction (Fig. 3)
. These data suggested that ritonavir inhibited the ability of docetaxel to induce expression of CYP3A4at the transcriptional level.
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B in DU145 Cells.B stimulates cell proliferation and confers cellular resistance to chemotherapy (15, 16, 17, 18)
. Therefore, NFB can be a molecular target of cancer treatment. The effect of ritonavir on NFB activity was explored using an ELISA-based assay. Control DU145 cells possessed strong NFB/DNA binding activity (data not shown). Treatment of DU145 cells with ritonavir (1 or 2 x 10–5 mol/L) inhibited the NFB binding activity by either 25 ± 7% or 53 ± 13%, respectively, as compared with untreated control cells (Fig. 4A)
. As control, 100-fold molar excess of the wild-type NFB consensus oligonucleotides was added to the assay of control lysate from untreated cells. Binding was inhibited by at least 80%; however, mutated NFB consensus oligonucleotides at the same molar excess were unable to inhibit binding (Fig. 4A)
, ascertaining the specificity of binding of NFB to its consensus binding site.
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B by siRNA Augmented Cytotoxicity of Docetaxel.B sensitizes DU145 cells to the antitumor effect of docetaxel, DU145 cells were transiently transfected with either NFB siRNA or control siRNA. After 48 hours, cells were harvested and subjected to Western blot analysis, showing that NFB siRNA effectively down-regulated levels of this transcription factor (Fig. 4B)
. Transfectants were cultured either with or without docetaxel for another 48 hours and subjected to MTT assay. NFB siRNA-transfected DU145 cells were more sensitive to growth inhibition mediated by docetaxel compared with nonspecific siRNA-transfected cells (Fig. 4C)
. For example, 10–10 mol/L docetaxel inhibited growth of control cells by 20%; however, it inhibited growth of NFB siRNA-transfected cells by 34% (P < 0.005) under similar culture conditions (Fig. 4C)
.
Ritonavir Increased the Antitumor Activity of Docetaxel In vivo.
We evaluated the ability of ritonavir to enhance the ability of docetaxel to inhibit the growth of AIPC DU145 cells growing as xenografts in male BNX nu/nu triple immunodeficient mice. One day after injection of DU145 cells, mice were treated with ritonavir, docetaxel, or both. Control mice received diluent alone. Tumor volume was measured every week (Fig. 5A)
, and tumor weights were determined at autopsy (Fig. 5B)
. Docetaxel markedly suppressed the growth and weights of DU145 tumors. No statistical significance was noted in either the size or weights of the DU145 tumors in the mice that received ritonavir alone as compared with the control mice; however, the combination of ritonavir and docetaxel significantly decreased the size (P = 0.05) of DU145 tumors compared with mice that received docetaxel alone (Fig. 5A)
. The difference of mean tumor weights between these two groups was even more significant (Fig. 5B
; P < 0.01). The tumors from mice that received docetaxel alone weighed 72 ± 41 mg. Conversely, tumors from mice that received the combination of docetaxel and ritonavir weighed only 27 ± 5 mg (Fig. 5B)
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. The mice that received ritonavir alone showed a similar histologic appearance as the control mice (Fig. 6B)
. The histologic analysis of the tumors from mice treated with docetaxel alone showed necrotic tissue with 40% of cancer cells undergoing necrosis (Fig. 6C)
. Cancer cells were not found in the mice that received the combination of docetaxel and ritonavir; the site of tumor was composed of necrotic and fibrotic tissues (Fig. 6D)
. Organs from mice treated with the various therapies did not show any changes compared with controls, including their liver, kidney, spleen, bone marrow, lung, and heart. During the study, all of the mice were weighed each week; the mean weights of each of the experimental groups were statistically the same as those of the control mice (data not shown). Blood analyses were performed several hours before the mice were sacrificed. No difference in the mean hematopoietic values and blood chemistries was observed between treated and untreated mice (data not shown).
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. Once tumor volume reached approximately a mean of 200 mm3, treatment was initiated. Ritonavir again significantly enhanced the ability of docetaxel to decrease tumor size (P = 0.01) and tumor weight (P = 0.008) of DU145 xenografts (Fig. 7A and B)
. We measured apoptosis of DU145 tumor cells using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. TUNEL-positive cells were negligible in control and ritonavir-treated mice. Conversely, docetaxel induced 8% of DU145 cells to become TUNEL positive. Importantly, when docetaxel was combined with ritonavir, TUNEL-positive cells increased to 16% (data not shown). At the end of this experiment, we sacrificed the mice, removed their tumors, extracted RNA, and measured levels of CYP3A4by real-time PCR. At 16 hours before sacrifice, either docetaxel or ritonavir alone or in combination was administered to the mice. As shown in Fig. 7C
, docetaxel increased expression of CYP3A4by approximately twofold in DU145 tumors; when docetaxel was combined with ritonavir, docetaxel-induced expression of CYP3A4 was blocked completely. We also extracted nuclear protein from tumors and explored the effect of ritonavir on NFB DNA binding activity (Fig. 7D)
. Control tumors possessed measurable NFB DNA binding activity (mean absorbance at 450 mm, 0.73 ± 0.15), and ritonavir inhibited this activity by 40% (mean absorbance at 450 mm, 0.43 ± 0.1). These results were consistent with those obtained from the in vitro studies (Figs. 3
and 4
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P-gp is an integral plasma membrane protein encoded by the multidrug-resistant (MDR) gene belonging to the ATP-binding cassette family of transporters (21) . It is an energy-dependent efflux pump for a wide variety of compounds, including anticancer drugs such as docetaxel (15) . The cancer cells from individuals with advanced, refractory cancers, including prostate, breast, and lung, overexpress P-gp transcripts (22 , 23) . Thus, overexpression of P-gp is considered to contribute to drug resistance. Ritonavir was shown to inhibit the activity of P-gp; thus, ritonavir also might enhance the activity of docetaxel by blocking its cellular efflux, although PIs, including ritonavir, also are substrate for P-gp (24 , 25) . We and other investigators found that P-gp–overexpressing doxorubicin-resistant breast cancer MCF-7 cells were resistant to docetaxel (data not shown; ref. 26 ). Our preliminary studies showed that ritonavir enhanced the ability of docetaxel to decrease the growth of these cells (data not shown).
Recent studies found that PIs inhibited 26S proteasome activity. Ritonavir and saquinavir inhibited the degradation of IB
, which prevented the nuclear translocation and transcriptional activation of NFB in Kaposi sarcoma and prostate cancer cells (5
, 7)
. This was associated with growth arrest and apoptosis of these cells. Cancer cells including prostate cancer often have hyperactivity of the NFB pathway, which can make these malignant cells relatively resistant to chemotherapy (16, 17, 18)
. In this study, we have found that DU145 cells possessed strong NFB DNA binding activity, and ritonavir decreased this activity in vitro (Fig. 4A)
and in vivo (Fig. 7D)
. This also may contribute to the increased cytotoxicity of the combination of ritonavir and docetaxel.
In summary, we suggest a novel approach to cancer therapy by using an active chemotherapeutic drug and a PI (ritonavir) to help reverse the mechanisms of drug resistance, including rapid drug metabolism, efficient removal of the drug from the target cancer cells, and down-regulation of the NFB pathway.
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FOOTNOTES |
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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.
Requests for reprints: Takayuki Ikezoe, Department of Internal Medicine, Kochi Medical School, Nankoku, Kochi, 783-8505, Japan. Phone: 81-88-880-2345; Fax: 81-88-880-2348; E-mail: ikezoet{at}med.kochi-ms.ac.jp
Received 8/27/03. Revised 7/21/04. Accepted 8/11/04.
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REFERENCES |
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gene in human malignancies. Cancer Res 2001;61:5307-10.B activation in human prostate cancer cells. Prostate 2002;52:183-200.[CrossRef][Medline]
B/IB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells. Cancer Res 2000;60:4426-32.This article has been cited by other articles:
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  Z. He, L. Qin, L. Chen, N. Peng, J. You, and X. Chen Synergy of Human Immunodeficiency Virus Protease Inhibitors with Chloroquine against Plasmodium falciparum In Vitro and Plasmodium chabaudi In Vivo Antimicrob. Agents Chemother., July 1, 2008; 52(7): 2653 - 2656. [Abstract] [Full Text] [PDF]
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A. Srirangam, R. Mitra, M. Wang, J. C. Gorski, S. Badve, L. Baldridge, J. Hamilton, H. Kishimoto, J. Hawes, L. Li, et al. Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer Clin. Cancer Res., March 15, 2006; 12(6): 1883 - 1896. [Abstract] [Full Text] [PDF]
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