Deguelin-Induced Inhibition of Cyclooxygenase-2 Expression in Human Bronchial Epithelial Cells
The increased expression of cyclooxygenase (COX)-2 significantly enhances carcinogenesis and inflammatory reactions, and its regulation may be a reasonable target for cancer chemoprevention. We demonstrated previously that deguelin inhibits proliferation of premalignant human bronchial epithelial (HBE) cells, such as 1799 cells and squamous HBE cells, by regulating phosphatidylinositol-3-kinase Akt activity, which is involved in COX-2 expression. We sought to determine the effect of deguelin on COX-2 expression in squamous HBE cells. Deguelin strongly inhibited COX-2 expression in squamous HBE cells, without affecting the COX-1 protein level. Deguelin inhibited proliferation of a variety of non-small cell lung carcinoma (NSCLC) cell lines through apoptosis and induced Bax expression in the H322 NSCLC and squamous HBE cells. Deguelin treatment did not affect Bcl-2 protein levels but increased expression levels of the proapoptotic protein p53 and the cyclin-dependent kinase inhibitors p21 and p27 in the squamous HBE cells. The sensitivity of the squamous HBE and NSCLC cells to deguelin and the inhibitory effects of deguelin on COX-2 expression in the squamous HBE cells indicate that regulation of COX-2 expression is involved in the chemopreventive action of deguelin in lung cancer.

title: 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
abstract: We previously showed that phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways cooperate to promote non–small cell lung cancer (NSCLC) cell proliferation in vitro. This study was designed to explore whether inhibition of these pathways effectively inhibits NSCLC tumor growth in vivo.  The effects of PI3K/Akt inhibitors {LY294002, adenoviruses expressing dominant-negative mutant of the p85α adaptor subunit of PI3K (Ad-dnp85α), dominant-negative Akt [Ad-HA-Akt(KM)], or PTEN (Ad-PTEN)}, MKK4/c-jun NH2-terminal kinase (JNK) inhibitor [SP600215, adenovirus expressing dominant-negative MKK4, Ad-MKK4(KR)], and their combinations on proliferation and apoptosis in NSCLC cells were tested in vitro and in vivo using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a flow cytometry-based terminal deoxynucleotidyl transferase–mediated nick-end labeling assay, Western blot and immunohistochemical analyses, and an NSCLC xenograft tumor model.  Ad-dnp85α significantly inhibited proliferation of a subset of NSCLC cell lines used in our study. Intratumoral injection of Ad-dnp85α induced a significant decrease in the growth of H1299 NSCLC xenograft tumors. Concurrent inhibition of the PI3K/Akt and MKK4/JNK pathways showed enhanced antiproliferative effects on H1299 cells in vitro and in vivo by increasing apoptosis.  PI3K/Akt and MKK4/JNK pathways cooperate to stimulate NSCLC cell proliferation by maintaining cell survival, suggesting that simultaneously targeting these two pathways might be an effective therapeutic strategy against NSCLC.

title: Molecular mechanisms of deguelin-induced apoptosis in transformed human bronchial epithelial cells.
abstract: Increasing evidence has demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway plays an important role in cell proliferation, apoptosis, angiogenesis, adhesion, invasion, and migration, functions that are critical to cancer cell survival and metastasis. Increased expression of activated Akt has been observed in the early stages of tobacco-induced lung carcinogenesis. Moreover, blocking the PI3K/Akt pathway specifically inhibits the proliferation of non-small cell lung cancer (NSCLC) cells, indicating that the PI3K/Akt pathway is a potential target for chemoprevention and therapy in lung cancer. The aim of this work is to study the lung cancer chemopreventive potential of PI3K/Akt inhibitors using an in vitro lung carcinogenesis model. We found that genetic or pharmacologic approaches targeting the PI3K/Akt pathway inhibited the proliferation of premalignant and malignant human bronchial epithelial (HBE) cells. After screening several natural products to identify a potential lung cancer chemopreventive agent, we have found that deguelin, a rotenoid isolated from Mundulea sericea (Leguminosae), specifically inhibits the growth of transformed HBE and NSCLC cells by inducing cell-cycle arrest in the G2/M phase and apoptosis, with no detectable toxic effects on normal HBE cells, most likely due to the agent's ability to inhibit PI3K/Akt-mediated signaling pathways. The specific sensitivity of premalignant and malignant HBE and NSCLC cells to deguelin suggests that this drug could be clinically useful for chemoprevention in early-stage lung carcinogenesis and for therapy in confirmed lung cancer.

title: Insulin-like growth factor binding protein-3 inhibits the growth of non-small cell lung cancer.
abstract: Insulin-like growth factors (IGFs) have mitogenic and antiapoptotic properties and have been implicated in the development of lung cancer. The effects of IGFs are modulated by insulin-like growth factor binding proteins (IGFBPs). This study explored the effects of IGFBP-3 on non-small cell lung cancer (NSCLC) cells after infection with an adenovirus constitutively expressing IGFBP-3 under the control of the cytomegalovirus promoter (Ad5CMV-BP3). We found that IGFs, especially IGF-I, stimulated the growth of NSCLC cells, and Ad5CMV-BP3 suppressed this IGF-I-induced NSCLC cell growth. We also found that the clonogenicity of H1299 cells in soft agar was markedly reduced by Ad5CMV-BP3. Furthermore, direct injection of Ad5CMV-BP3 into H1299 NSCLC xenografts s.c. established in athymic nude mice induced massive destruction of the tumors. Ad5CMV-BP3 did not induce detectable cytotoxicity on normal human bronchial epithelial cells, suggesting therapeutic efficacy of this virus. Ad5CMV-BP3 infection was accompanied by apoptotic cell death in vitro as detected by flow cytometry, DNA fragmentation analysis, and Western blot analysis on the expression of Bcl-2 and on the cleavage of poly(ADP-ribose) polymerase, a substrate of caspase 3. Immunofluorescence confocal microscopy was also used to show the apoptotic effect of Ad5CMV-BP3 in H1299 tumors established in nude mice. These findings indicated that IGFBP-3 was a potent inducer of apoptosis in NSCLC cells in vitro and in vivo. To delineate the underlying mechanism, we examined the effect of IGFBP-3 on Akt/protein kinase B and glycogen synthase kinase-3beta, downstream mediators of the phosphatidylinositol 3-kinase pathway, and on mitogen-activated protein kinase (MAPK), all three of which are activated by IGF-mediated signaling pathways and have important roles in cell survival. IGFBP-3 overexpression inhibited the phosphorylation of Akt and glycogen synthase kinase-3beta and the activity of MAPK. Furthermore, IGF-I rescued the NSCLC cells from serum depletion-induced apoptosis, and this rescue was blocked in Ad5CMV-BP-3-infected H1299 NSCLC cells. Transient transfection with activated Akt or constitutively active MAPK kinase-1, an upstream activator of MAPK, partially blocked IGFBP-3-induced apoptosis of NSCLC cells. These findings suggested that the growth-regulatory effect of IGFBP-3 on NSCLC cells was attributable in part to the inhibition of the IGF-induced survival pathway. These data demonstrate the importance of IGFBP-3 in the regulation of NSCLC cell proliferation, clonogenicity, and tumor growth, suggesting that IGFBP-3 is a target for the treatment of lung cancer and that Ad5CMV-BP3 is a potential therapeutic agent.

title: 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.
abstract: Overexpression of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in non-small-cell lung cancer (NSCLC) cells in vitro and in vivo. However, Ras-mediated signaling pathways could develop resistance to apoptotic activities of IGFBP-3 in NSCLC cells. We thus evaluated the therapeutic potential of the combination of IGFBP-3 and SCH66336, a farnesyltransferase inhibitor that blocks Ras activation, in NSCLC cell lines.



The effects of the combination of adenoviral IGFBP-3 (Ad-IGFBP3) and SCH66336 on proliferation and apoptosis of NSCLC cell lines (H1299, H596, A549, H460, H358, H322, and H226B) were assessed in vitro and in vivo by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometry-based terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, western blot analyses, and an NSCLC xenograft tumor model. The specific effects of Ad-IGFBP 3 and SCH66336 on mitogen-activated protein kinase and Akt were assessed by using adenoviral vectors that express constitutively active MEK1 or constitutively active Akt. Synergy was assessed by median effect analysis.



The combination of Ad-IGFBP3 and SCH66336 had synergistic antiproliferative effects in five cell lines (H1299, H596, A549, H460, and H322). Antiproliferative effects were accompanied by increased apoptosis in H460 cells in vitro. Overexpression of a constitutively active Akt but not a constitutively active MEK-1 rescued H460 cells from apoptosis induced by single or combined treatment of Ad-IGFBP3 and SCH66336. In H1299 tumor xenografts, Ad-IGFBP3 and SCH66336 was associated with decreased tumor volume, increased apoptosis, and decreased Akt levels.



The combination of Ad-IGFBP3 and SCH66336 decreased Akt expression and increased apoptosis in NSCLC cells in vitro and in vivo. Simultaneous treatment with IGFBP-3 and SCH66336 may have the potential to be an effective therapeutic strategy in NSCLC.

title: Effects of 9-cis-retinoic acid on the insulin-like growth factor axis in former smokers.
abstract: Insulin-like growth factor (IGF) axis has been associated with the risk of lung cancer. 9-cis-retinoic acid (9-cis-RA) has shown potential chemopreventive activities in former smokers. This study was designed to evaluate the effects of 9-cis-RA on IGF axis in former smokers to identify any benefit the retinoid may have in preventing lung cancer.



Serum concentrations of IGF-I, IGF binding protein (IGFBP)-3, and their molar ratio (IGF-I/IGFBP-3) were measured with radioimmunoassay kits in stored blood samples from the participants of an original chemoprevention trial. The participants had ceased smoking for at least 12 months and were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg) or placebo. All statistical tests were two-sided.



A total of 111 samples from the study's baseline and 84 samples from the 3 months treatment were analyzed. The serum concentrations of IGF-I and IGF-I/IGFBP-3 at baseline were significantly lower in female than in male participants. After 3 months of treatment, the serum level of IGF-I and IGF-I/IGFBP-3 were significantly lower in the 9-cis-RA group than in the placebo group (P = .03 and P < .01, respectively), but the IGFBP-3 level was significantly higher (P = .03).



9-cis-RA treatment modulated the IGF axis in former smokers, suggesting that the IGF axis is a potential target for the chemopreventive activities of 9-cis-RA and that the serum concentrations of IGF, IGFBP-3, and IGF-I/IGFBP-3 could serve as surrogate end point biomarkers of 9-cis-RA treatment.

title: A modified mini-primer set for analyzing mitochondrial DNA control region sequences from highly degraded forensic samples.
abstract: To facilitate the analysis of mitochondrial DNA (mtDNA) control region sequences from highly degraded skeletal remains, a modified mini-primer set was designed to overcome the limitations of the Armed Forces DNA Identification Laboratory (AFDIL) mini-primer set. This modified mini-primer set is less affected by nucleotide variability and PCR amplification conditions than the AFDIL mini-primer set, and was able to amplify the mtDNA sequences of 55-year-old skeletal remains with high efficiency, indicating that it is a useful tool for analyzing mtDNA control region sequences from highly degraded forensic samples.

title: Inducible nitric oxide synthase (iNOS) expression is increased in lipopolysaccharide (LPS)-stimulated diabetic rat glomeruli: effect of ACE inhibitor and angiotensin II receptor blocker.
abstract: Previously, we reported that high glucose enhanced cytokine-induced nitric oxide (NO) production by rat mesangial cells (MCs), and that the enhanced expression of the iNOS pathway may promote extracellular matrix accumulation by MCs. The present study was designed to examine whether the iNOS pathway is pathologically altered in experimental diabetic nephropathy, and whether therapy with angiotensin converting enzyme (ACE) inhibitor (imidapril: I) or angiotensin II type I receptor (AT1) blocker (L-158,809: L), ameliorates these changes. Male Sprague-Dawley rats were injected with diluent (control: C) or streptozotocin. At sacrifice after 4, 8 and 12 weeks, rats underwent either a 4 hour placebo or an intraperitoneal lipopolysaccharide (LPS, 2 mg/kg) challenge. Systolic blood pressure (SBP) and urinary protein excretion (UPE) increased significantly in diabetic (D) rats compared with C. The basal expression of glomerular iNOS mRNA was increased in D rats compared with that of C rats, by reverse- transcription (RT)-polymerase chain reaction (PCR), whereas there was no significant difference in the level of protein by Western blot analysis. Upon LPS stimulation, the iNOS mRNA and protein expression was significantly elevated in D rats. In D rats, this up-regulation, of LPS-stimulated iNOS expression, was equally ameliorated both by I and L in mRNA and protein levels. From immunohistochemistry (IHC), there was a negative staining for the iNOS within the glomeruli of five C rats without LPS treatment, but one of four rats, with LPS treatment, showed minimal iNOS staining in the glomeruli. In D rats, the glomerular mesangium and podocytes were positive for iNOS in each of three out of five rats with, and without, LPS treatment. In conclusion, LPS-stimulated glomerular iNOS expression was enhanced in diabetic pnephropathy, and the activation of angiotensin II may play a role in this enhancement.

title: Forkhead factor, FOXO3a, induces apoptosis of endothelial cells through activation of matrix metalloproteinases.
abstract: The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells (ECs). However, its effects on extracellular matrices (ECM), which are important in EC survival, remained unknown. Here, we evaluated the role of FOXO3a on EC-ECM interaction.



Constitutively active FOXO3a was transduced to human umbilical vein endothelial cells by adenoviral vector (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection led to dehiscence of ECs from fibronectin-coated plates, resulting in anoikis, which was significantly reversed by matrix metalloproteinase (MMP) inhibitor, GM6001. FOXO3a increased the expression of MMP-3 (stromelysin-1) but decreased the expression of tissue inhibitors of metalloproteinases-1 (TIMP-1), which was associated with increased MMP enzymatic activity in zymography. Pathophysiologic conditions such as serum starvation or heat shock also induced activation of endogenous FOXO3a, leading to activation of MMP-3 and apoptosis, which was reversed by GM6001. Delivery of Ad-TM-FOXO3a to the intraluminal surface in vivo led to EC denudation, disrupted vascular integrity, and impaired endothelium-dependent vasorelaxation.



Activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in ECs.

title: Trial of aripiprazole in the treatment of first-episode schizophrenia.
abstract: Aripiprazole is an atypical antipsychotic indicated for the treatment of adult patients with schizophrenia. It is effective and well tolerated in patients with schizophrenia or schizoaffective disorder. The aim of the present study was to investigate therapeutic efficacy and tolerability of aripiprazole in patients with first-episode schizophrenia.



Twenty-one patients meeting the DSM-IV criteria for schizophrenia were recruited. The Positive and Negative Symptom Scale (PANSS) and the Clinical Global Impressions Scale (CGI) were completed at the beginning of the study and again after 1, 2, 4, and 8 weeks of aripiprazole treatment. Side-effects were analyzed using the Udvalg for Kliniske Undersøgelser Side-Effect Rating Scale. Weight was checked at each testing session, and prolactin levels were measured at baseline and after 8 weeks of aripiprazole treatment.



Significant benefits were observed after the first week of treatment. After 1 week of aripiprazole treatment, subscale and total scores on the PANSS had decreased significantly. This significant decrease was maintained throughout the study period. The mean score of CGI severity was also significantly different after 2 weeks of aripiprazole administration when compared to baseline score, and the significance was maintained thereafter. Weight did not significantly change after aripiprazole administration. Although mean prolactin level was decreased at 8 weeks, the difference was not significant.



Aripiprazole is an effective and well-tolerated antipsychotic agent in patients with first-episode schizophrenia. Further investigations with larger samples are needed.

title: Catechol-O-methyltransferase Val158Met polymorphism affects therapeutic response to mood stabilizer in symptomatic manic patients
abstract: Catechol-O-methyltransferase (COMT) is a candidate gene for the pathogenesis of some psychiatric disorders. The aim of this study is to examine the role of the COMT gene Val158Met polymorphism on the clinical aspects of bipolar disorder including symptomatology and therapeutic response. This study comprised 144 unrelated manic patients who met strict DSM-IV criteria for bipolar I disorder and 157 healthy unrelated controls. All subjects were of Korean ethnicity. To evaluate the clinical symptoms, we used the Young Mania Rating Scale at baseline and 6 weeks after treatment. No statistically significant difference in genotype distribution was found between manic patients and normal controls. There was also no significant difference in symptomatology among the genotypes in manic patients. In therapeutic response, however, patients with the Met/Met genotype were significantly more frequent in the non-responder than in the responder group. Our results suggest that the COMT gene polymorphism in the therapeutic response to mood stabilizers in manic patients. Further studies with a larger number of subjects and well-controlled design will be required to better understand the role of the COMT gene polymorphism on the therapeutic response to mood stabilizer in manic patients.