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Tumor Biology |
Departments of Thoracic/Head and Neck Medical Oncology [H-Y. L., K-H. C., W. K. H., J. M. K.] and Genitourinary Medical Oncology [S. A. W., R. J. C.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Department of Pediatrics, Mattell Children’s Hospital, University of California, Los Angeles, California 90095-1752 [B. L., P. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence implicate IGFs and their receptor, IGF-1R, in many human malignancies, including carcinomas of the lung (3) . Two distinct signal transduction pathways have been identified for IGF-1R. One pathway activates Ras, Raf, and MAPK, the main mitogen-conducting pathway, and the other pathway involves PI3K, which is responsible for antiapoptotic signal transduction (4) . Therefore, we hypothesized that blocking the interaction of IGFs with IGF-1R or interrupting the signal transduction pathway of IGF-1R can abolish the mitogenic activity of IGFs on lung cancer.
IGFBP-3, one of six members of the IGFBP family, regulates bioactivity of IGFs by sequestering IGFs away from its receptor in the extracellular milieu, thereby inhibiting the mitogenic and antiapoptotic action of IGFs (5, 6, 7) . A negative correlation between serum IGFBP-3 levels and lung cancer risk (6) suggests a protective role of IGFBP-3 against the effects of systemic IGFs. IGFBP-3 also has IGF-independent antiproliferative and proapoptotic effects, as shown by the finding that IGFBP-3 overexpression inhibits the growth of IGF-1R null fibroblasts (7) . These effects of IGFBP-3 are probably mediated by other cell surface receptors, such as the TGF-ß receptor (8) . However, the intracellular mechanisms by which IGFBP-3 mediates its IGF-independent antiproliferative and proapoptotic effects remain largely unknown. It has been demonstrated that IGFBP-3 is translocated to the nucleus, where it could exert a direct influence on gene expression (9 , 10) ; thus, nuclear IGFBP-3 may mediate its IGF-independent cellular effects via direct or indirect interaction with growth-inhibitory genes, apoptotic genes, or both.
IGFBP-3 gene expression has been shown to be induced by growth-inhibitory (and apoptosis-inducing) agents such as TGF-ß1 (11)
, tumor necrosis factor-
(12)
, retinoic acid (13)
, anti-estrogen ICI 182780 (14)
, vitamin D and its analogues EB1089 and CB1093 (15)
, and the transcription factor p53 (16)
. Thus, these agents may mediate their cellular effects through IGFBP-3.
IGFBP-3 also can potentiate IGF bioactivity in several different cell types (17 , 18) . Although the mechanism for the enhancement of the activity of IGF is largely unknown, it is thought to occur after the association of IGFBP-3 with the cell membrane, thereby facilitating the binding of IGFs to its receptor (19) . Alternatively, surface-associated IGFBP-3 may be targeted for proteolysis into fragments that have reduced IGF affinity (20) . Whether IGFBP-3 assumes an inhibitory role or an enhancing role may depend on the cell type and the compound that induces its expression.
We investigated the effects of overexpression of IGFBP-3 on NSCLC cells using an adenoviral vector as a gene-delivery system. We demonstrated that infection with an adenovirus expressing IGFBP-3 under the control of the CMV promoter (Ad5CMV-BP3) inhibited the growth of NSCLC cells in vitro and in vivo by inducing apoptosis. We also found that IGFBP-3 is a potent inhibitor of the PI3K/Akt/PKB and MAPK signaling pathways, which are important mediators of cell survival. These findings suggest that IGFBP-3 is a potential target for lung cancer treatment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HA-MAPKK (MEK1/R4F) was a gift from Dr. Melanie Cobb (The University of Texas at Southwestern Medical Center, Dallas, TX).
Generation of Ad5CMV-BP3.
A full-length human IGFBP-3 cDNA was inserted into the 5' end of the bovine growth hormone polyadenylation signal at EcoRV of the pAd-shuttle vector. The IGFBP-3-containing shuttle vector was digested with BstB1/ClaI, inserted into the pAd-speed vector containing adenoviral DNA, and transfected into 293 cells. 293 cells were maintained in DMEM containing 10% FCS until the onset of the cytopathic effect. The presence of IGFBP-3 was confirmed by dideoxy-DNA sequencing and Western blot analysis. Viral titers were determined by plaque assays and spectrophotometric analysis. The pAd-shuttle vector and pAd-speed vector were kindly provided by Dr. Jack A. Roth (The University of Texas M. D. Anderson Cancer Center).
Western and Western Ligand Blot Analyses.
Conditioned media were collected from H1299 cells after adenoviral infection. Western blot and Western ligand blot analyses were performed using 30 µg of whole cell lysate or 30 µl of conditioned media as described previously (22)
.
Measurements of Cell Growth.
To measure the effects of IGF-I and IGF-II on proliferation of NSCLC cells, 2 x 103 NSCLC cell lines were incubated in serum-free medium containing 0.01–500 ng/ml IGF-I or IGF-II for 3 days. To measure the effects of Ad5CMV-BP-3 on proliferation of NHBE and NSCLC cell lines, these cells were seeded at 1 x 103 to 2 x 103 cells/well in 96-well plates. After 1 day, cells were untreated or infected with 1 x 103, 5 x 103, or 1 x 104 particles/cell of Ad5CMV-BP3 or Ad5CMV (parental virus) as a viral control. Infection was allowed to occur for 2 h in the absence of serum, and infected cells were grown in medium containing 100 or 250 ng/ml IGF-I. After 3 days of incubation, the growth of infected cells was measured by the MTT assay as previously described (22)
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Growth in Soft Agar.
The ability of NSCLC cells infected with Ad5CMV-BP3 to grow in anchorage independence was assessed in soft agar. Briefly, 1 x 103 H1299 cells were transduced with 1 x 103 or 1 x 104 particles/cell of Ad5CMV or Ad5CMV-BP3 for 1 day. They were then suspended in 1 ml of RPMI 1640 containing 10% FCS and 0.2% agarose and were plated in 12-mm tissue culture plates with 500 µl of RPMI 1640 containing 10% FCS and a 1% agarose underlay. After 7 days, cultures were resuspended in 0.2 ml of RPMI 1640 containing the same viruses at the same doses. Anchorage-independent growth was allowed to occur for 2 weeks, and colonies >125 µm in diameter were counted.
Inhibition of Tumor Growth in Vivo.
The effect of Ad5CMV-BP3 on established s.c. tumor nodules was determined in athymic nude mice in a defined pathogen-free environment. Briefly, mice were irradiated with 350 rad (137Cs source), and H1299 cells in 100 µl of complete medium were s.c. injected into the mice at a single dorsal site. After the tumor volume reached 
75 mm3, 1 x 1010 viral particles of Ad5CMV-BP3 or Ad5CMV in 100 µl of 1x PBS, or 100 µl of 1x PBS alone as a control, was intratumorally injected. Tumor size and volume were measured every day for 17 days after injection. Mice showing necrotic tumors or tumors 
1.5 cm in diameter were euthanized. Results were expressed as the mean (±SD) tumor volume (calculated from five mice) relative to the tumor volume at the time of adenovirus injection (day 0).
Apoptosis Analysis.
Apoptosis was measured using the APO-BRDU staining kit (Phoenix Flow Systems, San Diego, CA) as previously described (23)
. Briefly, H1299 cells were untreated or infected with Ad5CMV or Ad5CMV-BP3 and then allowed to grow in serum-free medium or medium containing 10% serum or 100 ng/ml IGF-I for 3 days. Floating and adherent cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) to determine the percentage of apoptotic cells. The percentage of dead cells was determined by FACS analysis of propidium iodide-stained nuclei. Apoptosis was also determined by detecting of nucleosomal DNA fragmentation, which was measured using the TACS apoptotic DNA laddering kit (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Briefly, DNA was isolated from untreated or virus-infected H1299 cells by incubating the cells in lysis buffer. DNA samples were then subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. To determine whether Ad5CMV-BP3-induced apoptosis was mediated through the inhibition of the PI3K/Akt/PKB and MAPK pathways, 2 x 105 H1299 cells were seeded onto six-well plates and transiently transfected with 2 µg of an expression construct containing constitutively active Akt (MyrAkt) or constitutively active MAPKK (MEK1/R4F) using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer’s protocol. After 4 h of transfection, H1299 cells were infected for 2 h with 1 x 104 particles/cell of Ad5CMV-BP3 or Ad5CMV as a control. Cells were changed to fresh RPMI 1640 containing 10% FCS and grown for 3 days. Apoptosis was measured using the APO-BRDU staining kit as described above.
Immunofluorescence Confocal Microscopy.
To determine whether Ad5CMV-BP3 induced IGFBP-3 expression and apoptosis in tumor nodules in nude mice, 1 x 1010 viral particles of Ad5CMV-BP3 or Ad5CMV were intratumorally injected as described above, and tumor tissues were collected from the mice 3 days later. Tissues were fixed with 10% formaldehyde and embedded in paraffin. Then, 5-µm-thick tumor tissue sections were analyzed for IGFBP-3 expression and apoptosis-induced DNA fragmentation. Briefly, sections were deparaffinized through a series of xylene baths and rehydrated through graded ethanol baths. Next, the sections were treated with 2.5% blocking serum to reduce nonspecific binding and were incubated with primary antihuman IGFBP-3 rabbit polyclonal antibody, 1:100 dilution, and followed by incubation with Fluorolink Cy3-labeled secondary antibody. Paraffinembedded tissue sections were analyzed for DNA fragmentation by TUNEL assay according to the manufacturer’s protocol (Roche Molecular Biochemicals). Samples were analyzed using an inverted confocal microscope (Zeiss, Inc., Jena, Germany) operated by KS400 software (Zeiss, Inc.).
Immune Complex Kinase Assay.
Immune complex kinase assays were performed as previously described (24)
. Briefly, untreated or virus-infected H1299 cells were grown for 2 days in RPMI 1640 containing 10% serum. After being washed with 1x PBS to eliminate residual serum, cells were serum starved for 1 day, activated by treatment with 50 ng/ml IGF-I for 15 min, and harvested using lysis buffer. Next, 100 µg of cell extracts were immunoprecipitated with a mixture of antibodies against p44 MAPK (ERK1)/p42 MAPK (ERK2) and with protein A-G agarose beads. After the beads were washed, an immune complex kinase assay was performed using MBP as a substrate.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. IGFBP-3 expression was not detectable in the cells incubated with medium alone or with Ad5CMV. The expression of IGFBP-3 peaked at day 3 and decreased at day 5. These results are consistent with those of previous reports that showed maximal adenovirus-mediated gene expression at day 3 and rapid decrease after day 5 (25)
. The expression pattern in other NSCLC cell lines infected with Ad5CMV-BP3 was consistent with that in H1299 cells (data not shown). The Western ligand blot analysis using conditioned media from H1299 cells infected with Ad5CMV-BP3 or with Ad5CMV indicated that IGFBP-3 secreted from Ad5CMV-BP3-infected H1299 cells bound strongly to IGF-I (Fig. 1B)
and IGF-II (data not shown).
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, showing the mitogenic effect of IGF-I on NSCLC cells. IGF-II showed a minimal mitogenic effect on NSCLC cell growth (data not shown). Hence, we investigated the effects of Ad5CMV-BP3 on IGF-I-stimulated NSCLC cell growth. Relative to the control, Ad5CMV-BP3 infection inhibited IGF-I-induced growth of NSCLC cells in a viral dose-dependent manner. In contrast, Ad5CMV-BP3 showed minimal inhibitory effects on NSCLC cell growth in the absence of IGF-I. Importantly, Ad5CMV-BP3 was not detectably cytotoxic to NHBE cells regardless of the stimulation of IGF-I (Fig. 2B)
. Previous studies have suggested that IGF-1R is particularly important in anchorage-independent growth and that its inhibition also suppresses tumorigenicity (26
, 27)
. Therefore, anchorage-independent growth of H1299 cells infected with Ad5CMV-BP3 was assessed by counting colony formation in soft agar. Relative to the effect of controls, Ad5CMV-BP3 infection prominently inhibited colony formation of H1299 cells in soft agar, with a 90% decrease in cells infected with 1 x 104 particles/cell of Ad5CMV-BP3 (Fig. 2C)
. These findings suggested that Ad5CMV-BP3 is capable of suppressing the tumorigenicity of H1299 NSCLC cells.
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. DNA fragmentation analysis on H1299 cells infected with 1 x 104 particles/cell of Ad5CMV-BP3 showed DNA ladders (Fig. 4B)
. Because the Bcl protein family has an important role in apoptosis, we examined the level of Bcl-2 and Bax in Ad5CMV-BP3-infected H1299 cells using Western blot analysis. Ad5CMV-BP3 inhibited the expression of Bcl-2 in a dose-dependent manner without changing the level of Bax, suggesting a role of IGFBP-3 in modulating the Bax:Bcl-2 ratio (Fig. 4C)
. Western blot analysis was also performed to determine whether Ad5CMV-BP3 induced a loss of the caspase-3 proenzyme (Mr 32,000) and cleavage of PARP, a substrate of caspase-3 proteolysis. H1299 cells infected with 1 x 104 particles/cell of Ad5CMV-BP3 for 3 days showed a significant decrease in the Mr 32,000 caspase-3 proenzyme and in induction of the Mr 89,000 fragment of PARP cleaved from the Mr 113,000 form of PARP. These findings indicate that IGFBP-3 is a potent inducer of apoptosis in NSCLC cell lines. To further investigate whether induction of IGFBP-3 caused apoptosis in vivo, immunofluorescence analysis and TUNEL assay were performed on tumors that were removed 3 days after a single administration of adenovirus. Compared with the effect of Ad5CMV, Ad5CMV-BP3 injection markedly increased IGFBP-3 and TUNEL staining (Fig. 5)
, indicating that the expression of IGFBP-3 induced apoptosis in tumor tissues. Only minimal TUNEL staining occurred in the Ad5CMV-injected tumors, probably because of the toxicity of the empty virus. Confocal microscopy revealed that IGFBP-3 expression and DNA fragmentation were colocalized, indicating that the expression of IGFBP-3-induced apoptosis. Some regions showed strong TUNEL staining but no staining for IGFBP-3; this apoptosis may have been spontaneous or induced by IGFBP-3 that was degraded after induction of apoptosis.
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/ß at Ser-9/21 (28)
; therefore, we examined the levels of pAkt (Ser-473) and pGSK-3ß (Ser-9) as surrogates for PI3K activity. According to results of Western blot analysis and an immune complex kinase assay, treatment with 50 ng/ml IGF-I for 15 min increased the levels of pAkt and pGSK-3ß and induced MAPK activity in H1299 cells (Fig. 6, A and B
, left). Ad5CMV-BP3 inhibited IGF-I-induced phosphorylation of Akt (Ser-473) and GSK-3ß (Ser-9) in H1299 cells in a dose-dependent manner, whereas the levels of pAkt (Ser-473) and pGSK-3ß (Ser-9) were not affected by IGF-I in untreated and Ad5CMV-infected H1299 cells (Fig. 6A)
. The total protein levels of Akt and GSK-3ß were not changed by these treatments.
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, suggesting that IGFBP-3 suppressed the IGF-I-induced activation of the PI3K/Akt/PKB and MAPK pathways in H1299 cells.
Overexpression of Constitutively Active Akt or MEK1 Protects H1299 Cells from IGFBP-3-induced Apoptosis.
We investigated whether IGFBP-3 inhibits IGF-induced cell survival pathways in NSCLC cells. Evidence of apoptosis was assessed in H1299 cells, which were untreated or infected with Ad5CMV or Ad5CMV-BP3 and then allowed to grow in the absence or presence of IGF-I. According to flow cytometric analysis, IGF-I treatment rescued H1299 cells from serum depletion-induced apoptosis in H1299 NSCLC cells. However, this rescue was blocked by the overexpression of IGFBP-3 (Fig. 7A)
, suggesting that IGFBP-3 interferes with survival function of IGF-I. To further explore whether IGFBP-3-induced apoptosis was mediated through the inhibition of IGF-I-induced signaling pathways, the susceptibility to the induction of apoptosis by Ad5CMV-BP3 was assessed in H1299 cells transfected with constitutively active Akt (MyrAkt) or constitutively active MEK1 (R4F) followed by the infection with Ad5CMV-BP3 or Ad5CMV. The equal transfection in each condition was verified by Western blot analysis on MyrAkt and MEK1 (data not shown). According to flow cytometry analysis, 10.4% of MyrAkt-transfected H1299 cells and 27.9% of MEK1-transfected cells showed induction of apoptosis by 1 x 104 particles/cell of Ad5CMV-BP3, as compared with 43% of induction in pCMV (empty vector)-transfected cells (Fig. 7B)
, suggesting that the induction of apoptosis by IGFBP-3 in NSCLC cells was attributable in part to the inhibition of the IGF-induced PI3K/Akt/PKB and MAPK pathways. Taken together, these results suggested a crucial role of IGFBP-3 in PI3K/Akt/PKB and MAPK-mediated cell survival pathways.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After we confirmed the function of Ad5CMV-BP3 in induction of IGFBP3 expression by Western blot and in binding of secreted IGFBP-3 to IGF-I and IGF-II by Western ligand blot analysis, we investigated the growth inhibitory effects of IGFBP-3. IGFs stimulated the growth of a subset of NSCLC cell lines, a result in agreement with previous findings (30) that showed a 1.1–2.7-fold increase in cell number stimulated by IGF-1 in a panel of NSCLC lines. The effects of IGF-I on NSCLC cell growth were relatively modest, with the exception of H358 cells, compared with the values reported for other models. This may be related to the fact that lung tumor cells with functional IGF receptors are able to enhance their own growth by synthesis of endogenous IGFs (31) . Thus, exogenous IGFs may induce only a modest increase of NSCLC cell growth. Ad5CMV-BP3 inhibited the IGF-I-stimulated growth of these cells, which is consistent with previous findings that showed an IGF-dependent, growth-inhibitory effect of IGFBP-3 in human breast cancer cells (27) . However, Hochscheid et al. (32) found that IGFBP-3 induced IGF-independent growth inhibition in a NSCLC cell line, observing that a higher concentration of IGF-I did not influence the proliferation of NSCLC cells stably transfected with IGFBP-3. The difference in these results might have been attributable to cell type specificity or different model systems used in the studies. In addition, stable transfection of IGFBP-3 might cause IGF-independent cell growth, and the growth of stably transfected cells could be regulated directly by IGFBP-3.
The crucial roles of IGF-1R in the establishment and maintenance of the transformed phenotype have been underscored (23 , 28 , 29) . In our study, infection of Ad5CMV-BP3 decreased the clonogenicity of H1299 cells in soft agar by >90%, which was comparable with the 84% decrease by an adenovirus expressing antisense IGF-1R (23) . Furthermore, the growth of NSCLC tumors established in nude mice was decreased by the injection of Ad5CMV-BP3, indicating that IGFBP-3 overexpression inhibits the growth of NSCLC cells in vitro and in vivo. These dramatic effects of IGFBP-3 could be caused by combined IGF-dependent suppression of IGF-1R signaling and direct IGF-independent effects, possibly mediated via retinoid X receptor (9) .
IGFs are known to protect cells from a variety of apoptotic stimuli through an IGF-1R-mediated cell survival pathway. Studies have shown that overexpression of IGF-1R protects cells against apoptosis, that suppression of IGF-1R induces apoptosis (5) , and that the sequestration of IGFBP-3 of IGFs away from IGF-1R induces apoptosis (5, 6, 7 , 16) . On the basis of these results, we hypothesized that IGFBP-3 acts as an effector of apoptosis in NSCLC cells by inhibiting IGF-I-mediated cell survival, antiapoptotic pathways, or both. In fact, we found evidence showing that Ad5CMV-BP3 induced apoptosis in NSCLC cells in vitro and in vivo, and that activity of Akt/PKB and MAPK, mediators of IGF-induced signaling pathways, were suppressed by overexpression of IGFBP-3. It has been demonstrated that Akt/PKB and MAPK have potent inhibitory effects on apoptosis (33) . Activation of IGF-1R by IGFs has been associated with an abrogation of caspase-3, caspase-7, and caspase-1 activity and changes in both proapoptotic and antiapoptotic members of the Bcl-2 family in a variety of cell systems (33 , 34) . The serine-threonine protein kinase Akt, a downstream mediator of the PI3K pathway, is also involved in phosphorylation of the proapoptotic protein BAD, releasing BAD from heterodimerization with cytoprotective proteins, such as Bcl-2 and Bcl-xL, thereby inhibiting its proapoptotic activity (4 , 33 , 34) . It is plausible that the IGF-dependent effects of IGFBP-3 reverse these actions and that IGFBP-3 therefore acts as an effector of apoptosis. We found that apoptosis induced by serum depletion of H1299 NSCLC cells were rescued by IGF-I treatment. However, this rescue was blocked by overexpression of IGFBP-3. Furthermore, constitutively active Akt or constitutively active MAPKK partially blocked the apoptosis induced by Ad5CMV-BP3, suggesting that IGFBP-3 induces apoptosis in NSCLC cells, in part via inhibition of the IGF-I-induced PI3K/Akt/PKB and MAPK pathways. This report is the first to show that IGFBP-3 induces apoptosis in NSCLC cells via inhibition of the signal transduction mechanism involved in cellular proliferation and survival.
The role of IGFBP-3 as an inhibitor of Akt/PKB activation has particular clinical implications, especially in the treatment of NSCLC, where constitutive activation of Akt/PKB occurs at a high frequency (35) . The role of Akt/PKB in survival has been demonstrated in studies in which cells were exposed to different apoptotic stimuli such as UV irradiation, growth factor withdrawal, cell cycle discordance, DNA damage, and TGF-ß (36, 37, 38, 39) . Manipulating Akt/PKB activity alters the sensitivity of cells to chemotherapy and irradiation; addition of a PI3K inhibitor or transfection of kinase-dead Akt/PKB into cells with high levels of Akt/PKB activity causes dramatic sensitization to these treatments (35) . Therefore, targeting Akt/PKB by using Ad5CMV-BP3 can enhance the efficacy of chemotherapy and radiation therapy and increase the apoptotic potential of NSCLC cells.
In conclusion, our study produced several important findings:
(a) We demonstrated that delivery of IGFBP-3 via a recombinant adenovirus inhibits lung cancer cell growth in vitro and reduces tumor growth in vivo via induction of apoptosis.
(b) We demonstrated that Ad5CMV-BP3 has no cytotoxicity on NHBE cells, suggesting a therapeutic potential of IGFBP-3. However, the nonspecific toxicity demonstrated by reduction in cell number and agar colonies at high titers of control virus (Ad5CMV) may be an obstacle to use Ad5CMV-BP-3 in clinical trials, despite the significant activity of Ad5CMV-BP3 in the regulation of cell growth.
(c) We demonstrated that adenoviral IGFBP-3 inhibits the MAPK and PI3K pathways, which have important roles in cell proliferation and survival. These results suggest that IGFBP-3 is an attractive target for lung cancer therapy and that Ad5CMV-BP3, as well as other therapeutic tools including small molecules targeting IGF-I:IGFBP-3 interaction, should be considered for clinical application. Important questions that remain are how to overcome the transient effect of adenoviral vector-induced gene expression, how to target more specifically to the site of tumors, and how to minimize the cytotoxicity of the adenoviral vector and the neutralizing effect of the antibodies that may arise in animals or patients after viral treatment.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Thoracic/Head and Neck Medical Oncology, Box 432, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-6363; Fax: (713) 796-8655; E-mail: hlee{at}mdanderson.org 
2 The abbreviations used are: NSCLC, non-small cell lung cancer; IGF, insulin-like growth factor; IGF-1R, IGF-1 receptor; IGFBP, IGF binding protein; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; TGF, transforming growth factor; CMV, cytomegalovirus; PKB, protein kinase B; NHBE, normal human bronchial epithelial; GSK, glycogen synthase kinase; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling; MBP, myelin basic protein; PARP, poly(ADP-ribose) polymerase; FACS, fluorescence-activated cell sorter.
Received 10/ 3/01. Accepted 4/12/02.
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H.-Y. Lee, S.-H. Oh, Y.-A. Suh, J. H. Baek, V. Papadimitrakopoulou, S. Huang, and W. K. Hong Response of Non-Small Cell Lung Cancer Cells to the Inhibitors of Phosphatidylinositol 3-Kinase/Akt- and MAPK Kinase 4/c-Jun NH2-Terminal Kinase Pathways: An Effective Therapeutic Strategy for Lung Cancer Clin. Cancer Res., August 15, 2005; 11(16): 6065 - 6074. [Abstract] [Full Text] [PDF]
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 X.-D. Wang Can Smoke-Exposed Ferrets Be Utilized to Unravel the Mechanisms of Action of Lycopene? J. Nutr., August 1, 2005; 135(8): 2053S - 2056S. [Full Text] [PDF]
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 H.-Y. Lee, Y. S. Chang, J.-Y. Han, D. D. Liu, J. J. Lee, R. Lotan, M. R. Spitz, and W. K. Hong Effects of 9-cis-Retinoic Acid on the Insulin-Like Growth Factor Axis in Former Smokers J. Clin. Oncol., July 1, 2005; 23(19): 4439 - 4449. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, H. Moon, K.-H. Chun, Y.-S. Chang, K. Hassan, L. Ji, R. Lotan, F. R. Khuri, and W. K. Hong Effects of Insulin-like Growth Factor Binding Protein-3 and Farnesyltransferase Inhibitor SCH66336 on Akt Expression and Apoptosis in Non-Small-Cell Lung Cancer Cells J Natl Cancer Inst, October 20, 2004; 96(20): 1536 - 1548. [Abstract] [Full Text] [PDF]
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 T. Ikezoe, S. Tanosaki, U. Krug, B. Liu, P. Cohen, H. Taguchi, and H. P. Koeffler Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid leukemia cells Blood, July 1, 2004; 104(1): 237 - 242. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, Y.-A. Suh, J. W. Kosmeder, J. M. Pezzuto, W. K. Hong, and J. M. Kurie Deguelin-Induced Inhibition of Cyclooxygenase-2 Expression in Human Bronchial Epithelial Cells Clin. Cancer Res., February 1, 2004; 10(3): 1074 - 1079. [Abstract] [Full Text] [PDF]
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 A. M. Mikheev, S. A. Mikheeva, B. Liu, P. Cohen, and H. Zarbl A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation Carcinogenesis, January 1, 2004; 25(1): 47 - 59. [Abstract] [Full Text] [PDF]
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 Y. Mao, S. Pan, S. W. Wen, and K. C. Johnson Physical Activity and the Risk of Lung Cancer in Canada Am. J. Epidemiol., September 15, 2003; 158(6): 564 - 575. [Abstract] [Full Text] [PDF]
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C. Liu, F. Lian, D. E. Smith, R. M. Russell, and X.-D. Wang Lycopene Supplementation Inhibits Lung Squamous Metaplasia and Induces Apoptosis via Up-Regulating Insulin-like Growth Factor-binding Protein 3 in Cigarette Smoke-exposed Ferrets Cancer Res., June 15, 2003; 63(12): 3138 - 3144. [Abstract] [Full Text] [PDF]
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K.-H. Chun, J. W. Kosmeder II, S. Sun, J. M. Pezzuto, R. Lotan, W. K. Hong, and H.-Y. Lee Effects of Deguelin on the Phosphatidylinositol 3-Kinase/Akt Pathway and Apoptosis in Premalignant Human Bronchial Epithelial Cells J Natl Cancer Inst, February 19, 2003; 95(4): 291 - 302. [Abstract] [Full Text] [PDF]
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Y. S. Chang, L. Wang, D. Liu, L. Mao, W. K. Hong, F. R. Khuri, and H.-Y. Lee Correlation between Insulin-like Growth Factor-binding Protein-3 Promoter Methylation and Prognosis of Patients with Stage I Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2002; 8(12): 3669 - 3675. [Abstract] [Full Text] [PDF]
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Y. S. Chang, K. Gong, S. Sun, D. Liu, A. K. El-Naggar, F. R. Khuri, W. K. Hong, and H.-Y. Lee Clinical Significance of Insulin-like Growth Factor-binding Protein-3 Expression in Stage I Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2002; 8(12): 3796 - 3802. [Abstract] [Full Text] [PDF]
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