Affiliations of authors: Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston; J. W. Kosmeder II, J. M. Pezzuto, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago.
Correspondence to: Ho-Young Lee, Ph.D., Box 432, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: hlee{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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Several natural compounds, especially plant products and dietary constituents, have been found to exhibit chemopreventive activities both in vitro and in vivo in model systems (1214). Their mechanisms of action vary widely, with many suppressing cell growth or modulating cell differentiation and a few also inducing apoptosis. Rotenoids, which constitute a class of compounds from the flavonoid family, have chemopreventive activity (15), act by inhibiting NADH:ubiquinone oxidoreductase activity and by suppressing steady-state mRNA levels and enzymatic activity of 12-O-tetradecanoylphorbol 13-acetate-induced ODC (15,16). One rotenoid, deguelin, has been isolated from several plant species, including Mundulea sericea (Leguminosae) (17). Deguelin has been shown to have cancer-chemopreventive effects in models of both skin and mammary tumorigenesis (18).
We used an in vitro lung carcinogenesis model system that consists of premalignant and malignant human bronchial epithelial (HBE) cells to evaluate the potential of deguelin as a chemopreventive agent against lung cancer. Here, we report on the effects and molecular mechanism of action of deguelin on HBE cells representing different stages of lung carcinogenesis.
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MATERIALS AND METHODS |
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Deguelin (Fig.1) was synthesized in four steps from the natural product rotenone (Sigma-Aldrich, Milwaukee, WI), as previously described (19). The final product was more than 98% pure. Deguelin was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10-3M and was stored in a nitrogen tank.
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Normal HBE (NHBE) cells were purchased from Clontech (Palo Alto, CA) and maintained according to the manufacturers recommended protocol. BEAS-2B cells, an HBE cell line immortalized with a hybrid adenovirus/simian virus 40 (20) (obtained from Dr. A. Klein-Szanto, Fox Chase Cancer Center, Philadelphia, PA), were previously used to derive both premalignant and malignant HBE cells (21) as follows. BEAS-2B cells were explanted, along with beeswax pellets or beeswax pellets containing cigarette smoke condensate (CSC), into rat tracheas that had been denuded of bronchial epithelium. The tracheas were then transplanted into the dorsal subcutaneous tissues of nude mice (21). Tumors developed after 6 months. From these tumors, a variety of cell lines were derived in vitro that exhibited different levels of tumorigenicity when transplanted into nude mice. For this study, we used three cell lines derived from these tumors, the characteristics of which have been described in detail (21,22). Two cell lines were premalignant: one (1799) was derived from BEAS-2B cells exposed to a beeswax control pellet, and one (1198) was derived from BEAS-2B cells exposed to a beeswax pellet containing CSC. The third cell line (1170-1) was malignant and was derived from BEAS-2B cells exposed to a beeswax pellet containing CSC. Although 1799 and 1198 cells are autonomous and grow indefinitely in culture, they are defined as premalignant because 1) neither was tumorigenic; 2) 1799 cell line was sensitive to serum in that it was growth inhibited and terminally differentiated into squamous cells similar to normal HBE and BEAS-2B cells; 3) in vitro invasiveness was detected after exposure of BEAS-2B cells (from which 1799 and 1198 cells were derived) to either phorbol myristate acetate or CSC; 4) in vitro exposure to the carcinogen N-nitrosamine-4-(methylnitrosamine)-1-(3 pyridyl)-1-butanone induced transformation of BEAS-2B cells and increased epidermal growth factor receptor (EGFR) expression in transformed BEAS-2B cells (21,23). Aberrant EGFR expression frequently occurs in bronchial preneoplasia, suggesting that squamous cell cancer development and EGFR expression are related (24). 1198 cells were not tumorigenic, although they were generated from CSC-treated BEAS-2B cells. The malignant 1170-1 cells were defined as a malignant cell line because they exhibit several features typical of invasive adenocarcinomas, including increased expression of EGFR and transforming growth factor- (TGF-
) (21). In addition, 1170-1 cells had acquired resistance to serum-mediated growth inhibition (21).
NHBE cells were induced to differentiate into squamous cells by growing them to confluence on tissue culture plates coated with a thin matrix of fibronectin (10 µg/mL; Upstate Biotechnology, Inc., Lake Placid, NY) and collagen (30 µg/mL; Celtrix Laboratories, Inc., Palo Alto, CA) as described (25). HB56B cells, an immortalized HBE cell line induced by loss of a portion of chromosome 11p without p53 or K-ras gene mutations (26) obtained from Dr. R. Reddel (National Cancer Institute, Bethesda, MD), NHBE cells, 1799 cells, and squamous HBE cells were grown in keratinocyte serum-free medium (KSFM; Life Technologies Inc., Gaithersburg, MD) containing EGF (2 µg/mL) and bovine pituitary extract (BPE, 25 µg/mL) (20). 1198 and 1170-1 cells were maintained in KSFM supplemented with 3% fetal bovine serum.
For the analysis of growth inhibition by deguelin, NHBE cells, HBE cell lines, and squamous HBE cells were cultured in KSFM containing EGF and BPE. To induce activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, H1299 non-small-cell lung cancer (NSCLC) cells, which were purchased from ATCC, and NHBE cells were cultured in the absence of serum and EGF for 1 day and then treated with 50 ng/mL insulin-like growth factor I (IGF-I) for 15 minutes.
Cell Treatment With Deguelin and Determination of Growth Inhibition
To measure the effects of deguelin on cell proliferation, NHBE, 1799, 1198, 1170-1, and HB56B cells were plated at concentrations of 2 x 103 to 4 x 103 cells/well in 96-well plates. The next day, cells were treated with either 0.1% DMSO as a diluent control or various concentrations of deguelin (final DMSO concentration = 0.1%). At the end of the assay time period, cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (27). Six replicate wells were used for each analysis, and data from replicate wells are presented as means with 95% confidence intervals (CIs). For some experiments, the drug concentration required to inhibit cell growth by 50% (IC50) was determined by interpolation from doseresponse curves. At least three independent experiments were performed.
Cell Cycle Analysis
NHBE and 1799 cells were plated at a concentration of 2 x 105 cells/well in six-well plates. The next day, cells were treated with deguelin (various concentrations) or DMSO (0.1%) for 3 days to achieve maximal antiproliferative effects (determined from growth curves). All cells (nonadherent and adherent) were harvested, fixed with 1% paraformaldehyde and 70% ethanol, stained with 50 µg/mL propidium iodide, and subjected to flow cytometric analysis to determine the percentage of cells in specific phases of the cell cycle (G1, S, and G2/M) as described (28). Flow cytometric analysis was performed using a Coulter EPICS Profile II flow cytometer (Coulter Corp., Miami, FL) equipped with a 488-nm argon laser. Approximately 10 000 events (cells) were evaluated for each sample. Two independent experiments were performed, and one is presented.
Apoptosis Assays
NHBE, premalignant (1799 and 1198), and malignant (1170-1) HBE (2 x 106) cells were exposed to various doses of deguelin or to DMSO (0.1%) for 3 days. Apoptosis was assessed by morphology, by a flow cytometry-based, modified terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and by the detection of fragmented DNA. For morphology, live cells were observed by light microscopy using a x100 objective. For TUNEL analysis, both adherent and nonadherent cells were harvested and pooled, fixed with 1% paraformaldehyde and 70% ethanol, and processed using the APO-BrdU staining kit (Phoenix Flow Systems, San Diego, CA), a modified TUNEL assay, as described (27). Cells treated with DMSO were used to gate the control nonapoptotic populations and as a reference for cells treated with deguelin. An internal control (HL-60 cells treated with camptothecin to induce apoptosis) provided in the apoptosis detection kit was also used to ensure that the TUNEL reaction was occurring during the staining procedure. For the detection of fragmented nucleosomal DNA, cells were processed using the TACS apoptotic DNA laddering kit (Trevigen Inc., Gaithersburg, MD), according to the manufacturers recommended protocol.
Immunoblotting
Whole cell lysates from 1 x 106 cells were prepared in lysis buffer as described (23). Equivalent amounts of protein were resolved by sodium dodecyl sulfate (SDS)polyacrylamide gels (7.5%12%) and transferred to a nitrocellulose membrane. After the membrane was blocked in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and 5% (w/v) nonfat powdered milk, the membrane was incubated with primary antibody at the appropriate dilution in TBS5% nonfat milk at 4 °C for 16 hours. The membrane was then washed multiple times with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The proteinantibody complexes were detected by enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL), according to the manufacturers recommended protocol.
The following antibodies and working dilutions were used for the western blots: rabbit polyclonal antibodies against human phosphorylated Akt (pAkt) (Ser473) (1 : 1000), Akt (1 : 1000), phosphorylated glycogen synthase kinase 3 (pGSK-3
) (Ser9) (1 : 1000), and mouse monoclonal antibody against human anti-phosphorylated MAPK (anti-pMAPK) (Thr202/Tyr204) (1 : 500) (Cell Signaling Technology, Beverly, MA); rabbit polyclonal anti-GSK-3
/
(1 : 1000) (BD Transduction Laboratories, Lexington, KY); rabbit polyclonal anti-Bax and anti-caspase-3 antibodies (1 : 2000) (Pharmingen, San Diego, CA); rabbit polyclonal anti-Bcl-2 (1 : 1000) and rabbit polyclonal anti-poly(ADP-ribose) polymerase (PARP) antibody (1 : 1000) (VIC 5; Roche Molecular Biochemicals, Indianapolis, IN); rabbit polyclonal anti-hemagglutinin (HA) antibody (1 : 1000), goat polyclonal antibodies against extracellular related kinase 1(Erk-1; 1 : 1000), Erk-2 (1 : 1000), and
-actin (1 : 4000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); rabbit anti-mouse immunoglobulin G (IgG)horseradish peroxidase conjugate (1 : 2000) (DAKO, Carpinteria, CA); and donkey anti-rabbit IgGhorseradish peroxidase conjugate (1 : 2000) and rabbit anti-goat IgGhorseradish peroxidase conjugate (1 : 2000) (Amersham Pharmacia Biotech, Arlington Heights, IL).
Immune Complex Kinase Assay for MAPK
Approximately 5 x 106 1799 cells were treated with 10-7M deguelin or 0.1% DMSO for various time periods in the absence of any additional stimulatory growth factors. The cells were then treated with 50 ng/mL IGF-I for 15 minutes to activate MAPK. Total cell extracts were prepared in lysis buffer (23), and ERK-1/2 or c-Jun N-terminal kinase (JNK) were immunoprecipitated from 100 µg of each cell extract using antibodies (1 µg) that recognize ERK1/2 or JNK (Santa Cruz Biotechnology) and protein-A sepharose beads (20 µL) (Amersham Pharmacia Biotech). Kinase assays were performed by incubating the beads with 30 µL of kinase buffer to which 5 µCi of [32P]ATP (2000 cpm/pmol) and 1 µg of substrate (myelin basic protein [MBP; Calbiochem, La Jolla, CA] or GSTcJun [179] [Santa Cruz Biotechnology]) were added, as previously described (27). The samples were suspended in Laemmli buffer, boiled for 5 minutes, and then analyzed by SDSpolyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Total cell lysates from Jurkat cells (Upstate Biotechnology, Inc.) and H1299 NSCLC treated with 50 ng/mL IGF-I for 15 minutes were used as positive controls. Changes in the level of phosphorylation in MBP or GST-cJun reflect changes in Erk1/2 and JNK activity, respectively.
PI3K Assay
Approximately 5 x 106 1799 cells were treated with 10-7M deguelin or 0.1% DMSO for different time periods (024 hours) in the absence of any additional stimulatory growth factors. PI3K in these cells was then activated by treatment with 50 ng/mL IGF-I for 15 minutes. Cells were lysed, and PI3K was immunoprecipitated from 500 µg of total cell extracts with 5 µL of rabbit antibody against full-length rat p85 PI3K (Upstate Biotechnology, Inc.), which coprecipitates the p110 catalytic subunit of PI3K, and 20 µL of protein Asepharose beads (Amersham Pharmacia Biotech). PI3K activity in the immunoprecipitates was analyzed using bovine brain extract (Type I; Sigma-Aldrich), which contains a mixture of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate as a substrate, as described (29). Jurkat cell lysates (Upstate Biotechnology, Inc.) and IGF-I-activated H1299 NSCLC cell lysates were used as positive controls.
Generation of Adenoviral Vectors and Cell Infection Protocol
The pCMV6.Myr.Akt.HA vector plasmid and the vectors for adenovirus construction were kindly provided by Dr. Gordon Mills and Dr. Jack A. Roth (The University of Texas M. D. Anderson Cancer Center, Houston), respectively. Ad5CMV (parental virus) was used as a viral control. An adenoviral vector expressing a full-length Akt (also known as human protein kinase B) with an Src myristoylation signal fused in-frame to the c-Akt coding sequence, and an HA epitope (MyrAkt-HA) (29) under the control of cytomegalovirus (CMV) promoter (Ad5CMV-MyrAkt-HA) was constructed using the pAd-shuttle vector system (27). Viral titers were determined by standard plaque assays and spectrophotometric analysis of DNA content. The presence of MyrAkt-HA in viral DNA was confirmed by DNA sequencing of the vector.
Cells were untreated or infected with either Ad5CMV-MyrAkt-HA or Ad5CMV as a viral control. Infection was allowed to occur for 2 hours in the absence of serum, and then the infected cells were suspended in fresh medium. After 3 days of incubation, the induced expression of MyrAkt-HA in 1799 cells and squamous HBE cells by the adenoviral vector was examined by western blot analysis for Akt and HA. The function of Ad5CMV-MyrAkt-HA was examined by a western blot analysis of cell lysates for pGSK-3 (Ser9), which is a downstream target of Akt.
To determine whether deguelin-induced antiproliferative effects on premalignant HBE cells were mediated through the inhibition of the PI3K/Akt pathway, 2 x 105 1799 cells/well or 1 x 106 squamous HBE cells/well in six-well plates were infected with 5 x 103 particles/cell of a control virus (Ad5CMV) or with 1 x 103 or 5 x 103 particles/cell of Ad5CMV-MyrAkt-HA, an adenoviral vector that expresses a constitutively active Akt (MyrAkt) in KSFM. After 1 day of infection, cells were treated with 10-7M or 10-6M deguelin, 2 x 10-6M or 4 x 10-6M N-(4-hydroxyphenyl)retinamide (4-HPR), or 0.1 % DMSO as a control and then incubated for 1 or 2 days. Apoptosis was analyzed using the APO-BrdU staining kit for TUNEL, western blot analyses for caspase-3, and the cleavage of PARP.
Northern Analysis
Approximately 1 x 107 to 2 x 107 NHBE cells and squamous HBE cells were lysed in 4 M guanidinium isothiocyanate, and total cellular RNA was extracted as described (22). RNA (20 µg per sample) was electrophoresed through a 1% agarose gel containing 2% formaldehyde, transferred to a nylon membrane (Zeta-Probe; Bio-Rad Laboratories, Hercules, CA), and hybridized to a [-32P] dCTP (2'-deoxycytidine 5'-triphosphate)-labeled transglutaminase (TG) (30) or involucrine (Inv) (31) complementary DNA (cDNA), as described (31). Loading and integrity of each RNA sample was examined by observing the intensity of 18S and 28S in ethidium bromide-stained gels.
Statistical Analysis
Cell survival among groups was compared using Students t tests. All means and 95% CIs from triplicate samples were calculated using Microsoft Excel software (version 5.0; Microsoft Corporation, Seattle, WA). In all statistical analyses, two-sided P values of <.01 were considered statistically significant.
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RESULTS |
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To determine whether deguelin could be a potential lung cancer chemopreventive agent, we first examined its effects on the growth of normal, premalignant (1799 and 1198), and malignant (1170-1) HBE cells, which together constitute an in vitro progressive lung carcinogenesis model (21,22). We used a concentration range in vitro that was attainable in vivo. The growth of premalignant and malignant HBE cell lines was inhibited by deguelin in a dose- and time-dependent manner (Fig.2, A). After testing a range of concentrations from 10-9M to 10-7M, we determined that the IC50 for deguelin was less than 10-8M. Deguelin had minimal effect on the growth of NHBE cells. Of all the cell lines, premalignant 1799 cells, which represent the earliest stage in the lung cancer model, were the most sensitive to deguelin, with exposure to 10-7M deguelin for 1 day decreasing cell growth by 67.1% (95% CI = 64.1% to 70.1%). Because BEAS-2B cells have only a few of the properties of premalignant HBE cells in vivo, we also tested the effects of deguelin on cells from another immortalized cell line, HB56B. Dose- and time-dependent growth-inhibitory effects of deguelin in these cells were also detected (Fig.2
, A). These results suggest that deguelin preferentially inhibits growth of premalignant HBE cells.
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We assessed how deguelin affected cell growth by determining the effects of deguelin on the cell cycle by flow cytometry. Cell lines 1799 (Fig.2, B), 1198, and 1170-1 (data not shown) treated with deguelin (10-8M or 10-7M) for 3 days accumulated in the G2/M phase of the cell cycle. No detectable cell cycle changes were noted in NHBE cells treated with deguelin (10-7M) for 3 days.
Effects of Deguelin on Apoptosis In Vitro
Because cells that accumulate in the G2/M phase of the cell cycle often enter apoptosis, we hypothesized that deguelin may have inhibited growth by inducing apoptosis. In fact, cells that were treated with deguelin at greater than 10-8M for 1 day showed morphologic changes typical of apoptosis, including membrane blebbing, increased refractoriness, and chromatin condensation (data not shown). We confirmed that 1799 cells treated with deguelin were undergoing apoptosis by TUNEL staining and flow cytometry analysis (Fig.3, A). Although less than 1% of 1799 cells treated with DMSO underwent apoptosis, approximately 3.3% of 1799 cells treated with 10-9M deguelin, 68.5% of cells treated with 10-8M deguelin, and 92.2% of cells treated with 10-7M deguelin for 3 days underwent apoptosis (Fig.3
, A).
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We next assessed the expression of apoptosis-related enzymes (caspase-3 and PARP) and Bcl family members (Bcl-2, Bax, Bcl-xL). There was a decrease in the 32-kd caspase-3 proenzyme and a concomitant increase in the cleavage of the 113-kd fragment of PARP to the 89-kd form in 1799 cells treated with deguelin (10-8M or 10-7M) for 3 days, indicating that deguelin activated caspase-3 (Fig. 3, C). Deguelin also induced a dose-dependent increase in the level of Bax and a slight decrease in Bcl-2 expression in 1799 cells (Fig. 3
, C) but did not affect the level of Bcl-xL (Fig. 3
, C). We observed changes in the levels of these proteins in 1198 and 1170-1 cells treated with deguelin that were similar to the changes observed in 1799 cells (data not shown).
Effect of Deguelin on Components of MAPK and PI3K/Akt Signaling Pathways in Premalignant HBE cells
We investigated whether MAPK and PI3K/Akt, which are important in regulating cell apoptosis and proliferation (3235), were involved in deguelin-mediated apoptosis in 1799 cells. Because the activities of MAPK and Akt are regulated by phosphorylation, we examined the levels of phosphorylated MAPK (pP44/42 MAPK) and pAkt in NHBE, premalignant (1799 and 1198), and malignant (1170-1) HBE cells treated with deguelin for different time periods. Basal levels of unphosphorylated or phosphorylated MAPK in NHBE, 1799, 1198, and 1170-1 cells were similar (Fig.4, A). By contrast, basal levels of phosphorylated Akt were higher in premalignant (1799 and 1198) and malignant (1170-1) HBE cells than in NHBE cells (Fig.4
, A), although the levels of the unphosphorylated Akt and an unrelated protein (
-actin) were similar in these cells, suggesting that Akt was constitutively active in premalignant and malignant HBE cells. NHBE cells did not express a basal level of phosphorylated Akt; however, IGF-I could induce phosphorylation of Akt in NHBE cells (Fig.4
, B), indicating that the IGF-IR signaling pathway, which leads to Akt phosphorylation, is intact in NHBE cells.
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We next measured the effect of deguelin on PI3K activity. PI3K was immunoprecipitated with anti-p85 antibody from total cell extracts derived from the 1799 cells treated with 10-7M deguelin, activated with IGF-I, and tested in a kinase assay. Compared with PI3K activity from untreated cells, deguelin decreased PI3K activity approximately 55%; this decrease was not accompanied by decreased expression of the PI3K components (p85 and p110
) (Fig.4
, E). Total cell lysates from Jurkat cells and H1299 NSCLC cells treated with 50 ng/mL IGF-I for 15 minutes and from NHBE cells cultured in the absence of IGF-I activation were used as positive and negative controls, respectively. These findings indicated that deguelin appears to preferentially affect the PI3K/Akt signaling pathway in 1799 cells. Interestingly, the pAkt level was reduced relative to that in untreated cells after 7 hours of treatment (Fig.4
, D) and was virtually undetectable by 14 hours, although PI3K activity was still high during this time period (Fig.4
, E). This result suggests that deguelin may inhibit Akt activity through PI3K-independent pathways in addition to the PI3K-dependent pathway.
Effect of PI3K/Akt on Deguelin-Induced Death in Premalignant HBE Cells
To test the hypothesis that deguelin-induced apoptosis is mediated through the inhibition of the PI3K/Akt pathway, we constructed an adenovirus expressing a constitutively active form of Akt (Ad5CMV-MyrAkt-HA). We first tested its effects on endogenous Akt expression and activity in 1799 cells. Dose-dependent expression of the HA tag was detected in 1799 cells infected with Ad5CMV-MyrAkt-HA (Fig.5, A). Compared with 1799 cells infected with a control adenovirus (Ad5CMV), expression of MyrAkt-HA, which had a slower mobility (i.e., larger molecular weight) than endogenous Akt, did not affect levels of endogenous Akt. However, the level of pGSK-3
, a downstream Akt target, was increased in 1799 cells infected with Ad5CMV-MyrAkt-HA, thus indicating an increase in Akt activity in these cells. Next, we infected 1799 cells with Ad5CMV-MyrAkt-HA and tested them for susceptibility to treatment with deguelin. 1799 cells infected with Ad5CMV-MyrAkt-HA showed an increase in cell survival, relative to cells infected with Ad5CMV, in response to deguelin that was dependent on the viral load (Fig.5
, B). Compared with the growth of untreated control, growth of 1799 cells, which were uninfected or infected with 5 x 103 particles/cell of Ad5CMV and treated with 10-7M deguelin for 2 days, was decreased by 54% (95% CI = 52.2% to 56.2%). However, growth of 1799 cells infected with 5 x 103 particles/cell of Ad5CMV-MyrAkt-HA and treated with 10-7M deguelin was 85% (95% CI = 81.9% to 88.5%) that of control cell growth. Increasing the concentration of deguelin to 10-6M did not decrease the growth of 1799 cells infected with Ad5CMV-MyrAkt-HA.
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To determine whether expression of Ad5CMV-MyrAkt-HA in 1799 cells affects deguelin-induced apoptosis, we assessed apoptosis in 1799 cells infected with Ad5CMV-MyrAkt-HA and treated with deguelin. Treatment with deguelin (10-7M) induced apoptosis in approximately 40% of 1799 cells or 1799 cells infected with the control adenovirus but in less than 10% of 1799 cells infected with Ad5CMV-MyrAkt-HA (Fig.5, C), which suggests that the induction of apoptosis by deguelin in 1799 cells results, at lease in part, from an inhibition of the PI3K/Akt-mediated anti-apoptotic pathway.
Effects of Deguelin on Squamous HBE Cells
The premalignant and malignant cell lines used in this study were derived from an HBE cell immortalized with a hybrid adenovirus/simian virus 40 (21). Adenovirus interaction with v integrins, an event required for adenovirus internalization, also activates PI3K (38). Thus, we sought to confirm that the increased level of pAkt in 1799 cells was related to the stage of disease and was not an artifact of the cell lines origin by assessing the level of pAkt in squamous HBE cells. These cells mimic bronchial metaplasia, a potentially premalignant lesion induced by tobacco smoke (25).
Squamous HBE cells express higher levels of TG and Inv than NHBE cells (25). To confirm that we had indeed induced squamous HBE cells by culturing them on fibronectin and collagen, we assessed expression of TG and Inv in NHBE and squamous HBE cells by northern blot analysis and found that both mRNAs were expressed (Fig.6, A, left). We next examined expression of pAkt and pGSK-3
in squamous HBE cells to determine whether the PI3K/Akt pathway was constitutively active in these cells. Although the expression of unphosphorylated Akt and GSK-3
/
was similar in NHBE and squamous HBE cells, the level of pAkt and pGSK-3
was markedly higher in squamous HBE cells than in NHBE cells (Fig.6
, A, middle), suggesting that the PI3K/Akt pathway was activated in squamous HBE cells. We then examined whether deguelin would inhibit PI3K/Akt activity in squamous HBE cells. Treatment with deguelin decreased the levels of pAkt and pGSK-3
in a time-dependent manner (Fig.6
, A, right).
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To test whether deguelin induced apoptosis in squamous HBE cells by inhibiting the PI3K/Akt pathway, squamous HBE cells were infected with Ad5CMV or Ad5CMV-MyrAkt-HA and were treated with deguelin (10-7M or 10-6M) for 1 day. Deguelin decreased proliferation of control squamous HBE cells in a dose-dependent manner (Fig.6, D). However, the growth of squamous HBE cells that overexpressed constitutively active Akt and were treated with deguelin (10-7M to 10-6M) for 1 day was approximately 95% (95% CI = 92.8% to 96.6%) of the growth of untreated cells (Fig.6
, D). In addition, deguelin induced a loss of caspase-3 and a concomitant increase in PARP cleavage in squamous HBE cells (control or infected with Ad5CMV), but it induced an increase in caspase-3 and a decrease in PARP cleavage in squamous HBE cells infected with Ad5CMV-MyrAkt-HA (Fig.6
, E), indicating that deguelin induction of apoptosis in squamous HBE cells involved inhibition of the PI3K/Akt pathway.
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DISCUSSION |
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Our results demonstrate that deguelin inhibited the growth of premalignant and malignant HBE cells, with minimal effects on the growth of NHBE cells. The premalignant cell line 1799 was the most sensitive of the lines tested to deguelin-induced antiproliferative effects, indicating that deguelin may have potential as a chemopreventive agent against lung cancer. Premalignant and malignant HBE cells treated with deguelin accumulated in the G2/M phase of the cell cycle and underwent apoptosis in a dose- and time-dependent manner. We found that deguelin increased the expression of Bax and decreased the expression of Bcl-2 in premalignant and malignant HBE cells, suggesting that changes in the ratio of Bax to Bcl-2 might contribute to the apoptosis-promoting activity of deguelin in these cells. However, changes in expression levels of Bcl family members were also observed in malignant HBE cells treated with deguelin, suggesting that other mechanisms may contribute to the sensitivity of premalignant HBE cells to deguelin.
In our ongoing efforts to determine the mechanism that mediates the effects of deguelin on premalignant HBE cells, we studied the involvement of the PI3K/Akt pathway. The PI3K/Akt pathway has been implicated in the development of multiple human cancers (4043). The isolation of a retroviral oncogene, v-p3k, coding a homologue of the PI3K p110 catalytic subunit, established that PI3K has an active role in oncogenic transformation (40). PIK3CA, the alpha catalytic subunit of PI3K, encoding p110, is amplified in human ovarian cancer cell lines (41), and an oncogenic mutant of p85 can transform mammalian fibroblasts when introduced with the v-raf oncogene (42). In addition, mammalian fibroblasts transfected with a constitutively active form of p110
have a partially transformed phenotype (43). Akt, an important and probably essential downstream component of PI3K-mediated oncogenic signaling (4244), provides a critical cell survival signal for tumor progression by phosphorylating a number of proteins involved in cell cycle regulation and pro-apoptotic factors (27,4345).
We found that Akt is constitutively active in premalignant and malignant HBE cells but not in NHBE cells. Because Akt activity is higher in 1799 cells (an immortalized HBE cell line) than in 1198 cells (a carcinogen-exposed immortalized HBE cell line) or in 1170-1 cells (a malignant HBE cell line), we suggest that Akt activation is an early event during lung carcinogenesis. Akt overexpression has been observed in early sporadic colon carcinogenesis (46), in normal ovarian surface epithelium from women with germline BRCA mutations (47), and in premalignant mammary hyperplasia, which has an increased risk of progressing to a tumor (48). These findings suggest that increased Akt activity resulting from Akt overexpression or constitutive activation from upstream molecules is a common feature during the early stages of carcinogenesis and that inhibition of Akt might be a potential target for a chemoprevention strategy.
Our data provide evidence that deguelin is a good potential chemopreventive agent, selectively blocking Akt activation and inducing apoptosis. The overexpression of constitutively active Akt blocked 1799 cells from undergoing deguelin-induced apoptosis, although it did not rescue these cells from 4-HPR-induced apoptosis, indicating that the inhibition of Akt activity by deguelin is an important mechanism of action in 1799 HBE cells. Exactly how deguelin inhibits Akt activity is unclear. Loss of Akt activity in response to deguelin was evident while substantial PI3K activity was still detectable, suggesting that deguelin inhibits Akt activity by a PI3K-independent mechanism. Indeed, Akt can be activated by a PI3K-independent mechanism that involves the Ca2+/calmodulin-dependent protein kinase kinase, in which the increase in the intracellular Ca2+ concentration promotes survival of some cultured neurons (49). We also observed that treatment of deguelin inhibits PI3K/Akt activity in 1198 and 1170-1 cells and that constitutively active Akt reduced the apoptotic effects of deguelin (data not shown). We examined whether this unique mechanism applied to other PI3K inhibitors and found that LY294002, a representiative PI3K inhibitor that blocks ATP binding to the p110 PI3K catalytic domain (50), was less effective than deguelin at inhibiting the growth of premalignant HBE cells (Lee HY: unpublished data); LY294002 concentrations higher than 10 µM were required to induce detectable growth inhibition in premalignant and malignant HBE cells, and those concentrations were cytotoxic to NHBE cells. The difference in efficacy between deguelin and LY294002 warrants investigation in view of the potential chemotherapeutic effect of deguelin.
Taken together, our results provide the first evidence, to our knowledge, that Akt is constitutively active in premalignant HBE cell lines and that deguelin can be a potential chemopreventive agent against lung cancer by targeting Akt activity. Deguelin blocks proliferation of premalignant and malignant HBE cells by inducing apoptosis. Deguelin is active at nanomolar levels, with no apparent cytotoxicity to normal HBE cells. Although rotenone is a pesticide, the U.S. Environmental Protection Agency, the National Center for Toxicological Research, and the National Toxics Rule have shown rotenone and other rotenoid-containing extracts to be noncarcinogenic and relatively nontoxic by oral administration (LD50 = 132 mg/kg in rats). A pharmacokinetic study in rats showed that a nontoxic dose of deguelin (1 µM), delivered intravenously or by oral gavage, is achievable in a variety of tissues in vivo, including lung tissue (51). Therefore, dosages of deguelin that induce apoptosis in vitro may be attainable in vivo in other models without potential side effects. Deguelin selectively blocks Akt activity in a PI3K-dependent or -independent manner, thereby attenuating the activity of a major anti-apoptotic pathway. The role of deguelin as an inhibitor of Akt activation also has clinical implications, especially in the treatment of NSCLC, in which constitutive activation of Akt occurs with high frequency (52). It has been reported that the manipulation of Akt activity, addition of a PI3K inhibitor, or transfection of kinase-dead Akt into cells with high levels of Akt activity dramatically increased sensitivity of NSCLC cells to chemotherapy and irradiation (52). Therefore, targeting Akt with deguelin may increase the efficacy of chemotherapy and radiation therapy and increase the apoptotic potential of NSCLC cells. How deguelin inhibits Akt activity in a PI3K-independent manner warrants further investigation.
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W. K. Hong is an American Cancer Society Clinical Research Professor.
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Manuscript received July 19, 2002; revised November 26, 2002; accepted December 16, 2002.
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