BPTF cooperates with p50 NF-κB to promote COX-2 expression and tumor cell growth in lung cancer.

Cyclooxygenase-2 (COX-2) is overexpressed in most human cancers, but its precise regulatory mechanism in cancer cells remains unclear. The aims of this study are to discover and identify the new regulatory factors which bind to the COX-2 promoter and regulate COX-2 expression and cancer cell growth, and to elucidate the mechanisms of action of these factors in lung cancer. In this study, the COX-2 promoter-binding protein BPTF (bromodomain PHD finger transcription factor) was detected, identified and verified by biotin-streptavidin-agarose pulldown, mass spectrum analysis and chromatin immunoprecipitation (ChIP) in lung cancer cells, respectively. The expressions of COX-2 and BPTF in lung cancer cell lines, mouse tumor tissues and human clinical samples were detected by RT-PCR, Western blot and immunohistochemistry assays. The interaction of BPTF with NF-kB was analyzed by immunoprecipitation and confocal immunofluorescence assays. We discovered and identified BPTF as a new COX-2 promoter-binding protein in human lung cancer cells. Knockdown of BPTF inhibited COX-2 promoter activity and COX-2 expression in lung cancer cells in vitro and in vivo. We also found that BPTF functioned as a transcriptional regulator through its interaction with the p50 subunit of NF-kB. Knockdown of BPTF abrogated the binding of p50 to the COX-2 promoter, while the inhibition of p50 activity abolished the decreased trend of COX-2 expression and lung cancer cell proliferation caused by BPTF silencing. Moreover, we showed that the expressions of BPTF and COX-2 in tumor tissues of lung cancer patients were positively correlated, and high co-expression of BPTF and COX-2 predicted poor prognosis in lung cancer patients. Collectively, our results indicated that BPTF cooperated with p50 NF-κB to regulate COX-2 expression and lung cancer growth, suggesting that the BPTF/p50/COX-2 axis could be a potential therapeutic target for lung cancer.

Synergistic Antitumor Effect of BKM120 with Prima-1Met Via Inhibiting PI3K/AKT/mTOR and CPSF4/hTERT Signaling and Reactivating Mutant P53.

BACKGROUND/AIMS: PI3KCA and mutant p53 are associated with tumorigenesis and the development of cancers. NVP-BKM120, a selective pan-PI3K inhibitor, exerts the antitumor activity by suppressing the PI3K signaling pathway. Prima-1Met, a low molecular weight compound, can rescue the gain-of-function of mutant p53 by restoring its transcriptional function. In this study, we investigated whether PI3K inhibition combined with mutant p53 reactivation could enhance the antitumor effect in thyroid cancer cells. METHODS: The effects of BKM120 and Prima-1Met on the proliferation, apoptosis, migration and invasion of thyroid cancer cells were measured by MTT, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Thyroid differentiation was assessed by detecting the expression levels of specific markers using RT-PCR and Western blot. The in vivo antitumor efficacy was analyzed in a mouse xenograft model. RESULTS: The combinational treatment of BKM120 and Prima-1Met significantly enhanced the inhibitions of cell viability, colony formation, migration and invasion, and the induction of apoptosis in thyroid cell lines, and synergistically suppressed tumor xenograft growth by inhibiting the PI3K/Akt/mTOR and EMT signaling pathways, up-regulating p53 targeted genes, and triggering the release of cytochrome c. Moreover, the combination of BKM120 and Prima-1Met suppressed the stemlike traits of thyroid cancer cells and promoted their differentiation by upregulating the expression of thyroid-specific differentiation markers and repressing the expression of cancer stem cell markers. Furthermore, the mechanism study demonstrated that the combinational treatment synergistically abrogated the binding of CPSF4 at the promoter of hTERT and thus suppressed hTERT expression. Consistently, overexpression of hTERT rescued the inhibitions of cell viability, invasion and stem-like traits mediated by the combination of BKM120 and Prima-1Met. CONCLUSION: Our results showed that the combination of BKM120 with Prima-1Met synergistically suppressed the growth of thyroid cancer cells and tumor xenografts via inhibiting PI3K/Akt/mTOR and CPSF4/hTERT signaling and reactivating mutant p53.

YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma.

Y-box binding protein 1 (YBX1) is involved in the multi-tumor occurrence and development. However, the regulation of YBX1 in lung tumorigenesis and the underlying mechanisms, especially its relationship with CDC25a, was remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and CDC25a in lung adenocarcinoma and identified their roles in the regulation of lung cancer growth. The retrospective analysis of 116 patients with lung adenocarcinoma indicated that YBX1 was positively correlated with CDC25a expression. The Cox-regression analysis showed only high-ranking TNM stage and low CDC25a expression were an independent risk factor of prognosis in enrolled patients. High expression of YBX1 or CDC25a protein was also observed in lung adenocarcinoma cells compared with HLF cells. ChIP assay demonstrated the binding of endogenous YBX1 to the CDC25a promoter region. Overexpression of exogenous YBX1 up-regulated the expression of the CDC25a promoter-driven luciferase. By contrast, inhibition of YBX1 by siRNA markedly decreased the capability of YBX1 binding to CDC25a promoter in A549 and H322 cells. Inhibition of YBX1 expression also blocked cell cycle progression, suppressed cell proliferation and induced apoptosis via the CDC25a pathway in vitro. Moreover, inhibition of YBX1 by siRNA suppressed tumorigenesis in a xenograft mouse model and down-regulated the expression of YBX1, CDC25a, Ki67 and cleaved caspase 3 in the tumor tissues of mice. Collectively, these results demonstrate inhibition of YBX1 suppressed lung cancer growth partly via the CDC25a pathway and high expression of YBX1/CDC25a predicts poor prognosis in human lung adenocarcinoma.

KLF4 downregulates hTERT expression and telomerase activity to inhibit lung carcinoma growth.

Krüppel-like factor 4 (KLF4) is a transcription factor that contributes to diverse cellular processes and serves as a tumor suppressor or oncogene in various cancers. Previously, we have reported on the tumor suppressive function of KLF4 in lung cancer; however, its precise regulatory mechanism remains elusive. In this study, we found that KLF4 negatively regulated hTERT expression and telomerase activity in lung cancer cell lines and a mouse model. In addition, the KLF4 and hTERT expression levels were significantly related to the clinicopathological features of lung cancer patients. Promoter reporter analyses revealed the decreased hTERT promoter activity in cells infected with Ad-KLF4, and chromatin immunoprecipitation analysis demonstrated that endogenous KLF4 directly bound to the promoter region of hTERT. Furthermore, the MAPK signaling pathway was revealed to be involved in the KLF4/hTERT modulation pathway. Forced expression of KLF4 profoundly attenuated lung cell proliferation and cancer formation in a murine model. Moreover, hTERT overexpression can partially rescue the KLF4-mediated suppressive effect in lung cancer cells. Taken together, these results demonstrate that KLF4 suppresses lung cancer growth by inhibiting hTERT and MAPK signaling. Additionally, the KLF4/hTERT/MAPK pathway is a potential new therapeutic target for human lung cancer.

BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas.

BPTF, a subunit of NURF, is well known to be involved in the development of eukaryotic cell, but little is known about its roles in cancers, especially in non-small-cell lung cancer (NSCLC). Here we showed that BPTF was specifically overexpressed in NSCLC cell lines and lung adenocarcinoma tissues. Knockdown of BPTF by siRNA significantly inhibited cell proliferation, induced cell apoptosis and arrested cell cycle progress from G1 to S phase. We also found that BPTF knockdown downregulated the expression of the phosphorylated Erk1/2, PI3K and Akt proteins and induced the cleavage of caspase-8, caspase-7 and PARP proteins, thereby inhibiting the MAPK and PI3K/AKT signaling and activating apoptotic pathway. BPTF knockdown by siRNA also upregulated the cell cycle inhibitors such as p21 and p18 but inhibited the expression of cyclin D, phospho-Rb and phospho-cdc2 in lung cancer cells. Moreover, BPTF knockdown by its specific shRNA inhibited lung cancer growth in vivo in the xenografts of A549 cells accompanied by the suppression of VEGF, p-Erk and p-Akt expression. Immunohistochemical assay for tumor tissue microarrays of lung tumor tissues showed that BPTF overexpression predicted a poor prognosis in the patients with lung adenocarcinomas. Therefore, our data indicate that BPTF plays an essential role in cell growth and survival by targeting multiply signaling pathways in human lung cancers.

Down-regulation of prostate stem cell antigen (PSCA) by Slug promotes metastasis in nasopharyngeal carcinoma.

Distant metastasis and local recurrence are still the major causes for failure of treatment in patients with nasopharyngeal carcinoma (NPC), making it urgent to further elicit the molecular mechanisms of NPC metastasis. Using a gene microarray including transcription factors and known markers for cancer stem cells, prostate stem cell antigen (PSCA) was found to be significantly down-regulated in metastatic NPC in lymph node, compared to its primary tumour, and in NPC cell lines with high metastatic ability compared to those with low metastatic ability. NPC patients with low PSCA expression had a consistently poor metastasis-free survival (p = 0.003). Knockdown and overexpression of PSCA respectively enhanced and impaired the migration and invasion in vitro and the lung metastasis in vivo of NPC cells. Mechanistically, the enhancement of NPC metastasis by knocking down PSCA probably involved epithelial-mesenchymal transition (EMT), by up-regulating N-cadherin and ZEB1/2 and by activating RhoA. The down-regulation of PSCA in NPC cells resulted directly from the binding of Slug to the PSCA promoter. PSCA may be a potential diagnostic marker and therapeutic target for patients with NPC.

Significant prognostic impact of chemoradiotherapy-induced hemoglobin decrease on treatment outcomes of nasopharyngeal carcinoma.

PURPOSE: To investigate prognostic impact of chemoradiotherapy-induced hemoglobin (Hb) decrease on treatment outcomes of endemic nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Eight hundred and fifteen non-metastatic NPC, receiving neoadjuvant chemotherapy followed by radiotherapy (NACT+RT group) or concomitant chemoradiotherapy (CCRT group), were enrolled in this study, who were regrouped according to pre-radiotherapy Hb (pre-RT Hb), post-radiotherapy Hb (post-RT Hb) and individual Hb decrease through radiotherapy or CCRT (△Hb), respectively. Survival curves were estimated using Kaplan-Meier method and compared by log-rank test. Multivariate analysis was performed using the COX proportional hazard model and binary logistic regression model. RESULTS: A poorer 5-year disease-free survival (DFS) was observed when pre-RT Hb<130.00 g/L. However, post-RT Hb<130.00 g/L was associated with significantly poorer 5-year locoregional recurrence-free survival (LRFS) (P=0.010) and disease specific survival (DSS) (P=0.008). Multivariate analysis with the COX proportional hazard model identified post-RT Hb<130.00 g/L as an independent negative prognostic factor for both LRFS (hazard ratio [HR], 1.896; 95% confidence interval [CI], 1.158-3.106; P=0.011) and DSS (HR, 1.767; 95% CI, 1.152-2.711; P=0.009). Similarly, △Hb <-15.00 g/L also predicted poorer 5-year LRFS (P=0.024) and DSS (P=0.015), which was confirmed in multivariate analysis as an independent adverse prognostic factor for LRFS (HR, 1.586; 95% CI, 1.058-2.377; P=0.026) and DSS (HR, 1.556; 95% CI, 1.087-2.227; P=0.016), respectively. Multivariate analysis with binary logistic regression model indicated that CCRT was a significantly independent predictor for post-RT Hb <130.00 g/L and △Hb < -15.00 g/L. CONCLUSIONS: Chemoradiotherapy-induced decreased Hb levels have negative influence on locoregional control and survival, and might counteract the benefit of neoadjuvant/concomitant chemotherapy. Further studies on supportive care to maintain sufficient Hb level during chemo-radiotherapy are warranted.

Flavokawain B inhibits the growth of acute lymphoblastic leukemia cells via p53 and caspase-dependent mechanisms.

The development of novel chemotherapeutic drugs is needed for the treatment of patients with acute lymphoblastic leukemia (ALL). In this study, the anti-leukemic effect and the potential molecular mechanisms of action of flavokawain B on ALL were investigated. Flavokawain B was found to significantly inhibit the cellular proliferation of B-ALL and T-ALL cell lines in a dose-dependent manner. It also induced cellular apoptosis by increasing the expression of p53, Bax and Puma, and activating the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, the enhancement of p53-dependent apoptosis by flavokawain B could be rescued by pifithrin-α, a pharmacological inhibitor of p53 transcriptional activity. Moreover, the proliferation of leukemia blast cells from 16 patients with ALL was inhibited by flavokawain B, and tumor growth in xenograft mice was also suppressed by this drug. In conclusion, our results demonstrate the therapeutic potential of flavokawain B for the treatment of ALL.

Paradoxical role of CBX8 in proliferation and metastasis of colorectal cancer.

The effect of polycomb chromobox (Cbx) proteins in cancer is context-dependent. The Chromobox homolog 8 (CBX8) was originally characterized as a transcriptional repressor, which inhibits cell proliferation in Ink4a-Arf-dependent and -independent manner. However, the role of CBX8 in colorectal cancer remains unknown. Here, we found that high CBX8 expression was associated with a low rate of distant metastasis and good prognosis in CRC patients, even though CBX8 was up-regulated in CRC cell lines and clinical samples. Knockdown of CBX8 inhibited CRC proliferation in vitro and in vivo, mostly by increasing p53 and its downstream effectors. However, knockdown of CBX8 enhanced CRC migration, invasion and metastasis in vitro and in vivo, in part through direct up-regulation of integrin ß4 (ITGB4) that in turn decreased RhoA activity. Collectively, the knockdown of CBX8 inhibited CRC proliferation, while promoting its metastasis, thus exerting paradoxical effects in CRC progression.

Targeting the anaphase-promoting complex/cyclosome (APC/C)- bromodomain containing 7 (BRD7) pathway for human osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in childhood and adolescence and has a propensity for local invasion and early lung metastasis. However, the current therapies often result in chemoresistance, and a therapeutic target is not available in the clinic for osteosarcoma. Here, we report that BRD7 forms a complex with the anaphase-promoting complex/cyclosome (APC/C) and is degraded by APC/C(cdh1) and APC/C(cdc20) during the cell cycle. Moreover, BRD7 is a tumor suppressor in osteosarcoma, and the BRD7 mutant resistant to degradation by APC/C is more efficient than the wild-type protein at suppressing proliferation, colony formation, and tumor growth of osteosarcoma in vitro and in vivo. The combination of proTAME, an inhibitor of APC/C, with chemotherapeutic drugs efficiently targets osteosarcoma in vitro. Furthermore, there is a strong inverse correlation of protein levels between BRD7 and Cdh1 or Cdc20, and lower BRD7 expression is an indicator for poor prognosis in patients with osteosarcoma. Collectively, our results indicate that targeting the APC/C-BRD7 pathway may be a novel strategy for treating osteosarcoma.

Glycogen synthase kinase-3ß, NF-κB signaling, and tumorigenesis of human osteosarcoma.

BACKGROUND: Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3ß in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. METHODS: We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3ß expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5-8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3ß in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3ß inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3ß activity with overall survival. RESULTS: Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3ß formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3ß had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3ß resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3ß resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3ß inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3ß, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3ß and NF-κB (109.2 months). CONCLUSION: GSK-3ß activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3ß and/or NF-κB pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma.

IL-24 gene transfer sensitizes melanoma cells to erlotinib through modulation of the Apaf-1 and Akt signaling pathways.

Interleukin-24 (IL-24) is a novel tumor suppressor/cytokine gene expressed in normal human melanocytes but for which expression is nearly undetectable in metastatic melanoma. Overexpression of the IL-24 protein has been shown to inhibit tumor cell proliferation and induce apoptosis in many melanoma cell lines, and is now considered a tumor suppressor. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been widely studied for the treatment of human lung cancer and other solid tumors, but the erlotinib-targeted therapy has not been tested in melanoma. The objective of this study is to investigate the potency of erlotinib in suppressing the growth of human melanoma cells and whether IL-24 could enhance the antitumor activity of erlotinib. In cell viability and apoptosis assays, treatment with erlotinib dependently inhibited the growth of different melanoma cell lines and when combined with adenoviral vector-mediated IL-24 gene therapy, a significant increase in cell growth inhibition and apoptosis induction resulted (P<0.05). Immunoblot assay showed that the combination treatment of erlotinib and IL-24 considerably increased the cleavage of caspase-3 and caspase-9 and the expression of Apaf-1 protein in melanoma cells, inducing activation of the Apaf-1-dependent apoptotic pathways. Moreover, this combination treatment markedly inhibited phosphorylation of the EGFR, phosphatidylinositol-3 kinase, and Akt proteins, inactivating the Akt-dependent cell survival signaling pathway. These results show that a combination of IL-24-mediated molecular therapy and EGFR inhibitors such as erlotinib may be a promising treatment strategy for human melanoma and will serve as a basis for guiding the combination treatment designs in future preclinical and clinical trials.

Tumor-specific activation of human telomerase reverses transcriptase promoter activity by activating enhancer-binding protein-2beta in human lung cancer cells.

The up-regulated expression and telomerase activity of human telomerase reverse transcriptase (hTERT) are hallmarks of tumorigenesis. The hTERT promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. However, little is known about how tumor cells differentially activate hTERT transcription and induce telomerase activity. In this study, we identified activating enhancer-binding protein-2beta (AP-2beta) as a novel transcription factor that specifically binds to and activates the hTERT promoter in human lung cancer cells. AP-2beta was detected in hTERT promoter DNA-protein complexes formed in nuclear extracts prepared only from lung cancer cells but not from normal cells. We verified the tumor-specific binding activity of AP-2beta for the hTERT promoter in vitro and in vivo and detected high expression levels of AP-2beta in lung cancer cells. We found that ectopic expression of AP-2beta reactivated hTERT promoter-driven reporter green fluorescent protein (GFP) gene and endogenous hTERT gene expression in normal cells, enhanced GFP gene expression in lung cancer cells, and prolonged the life span of primary lung bronchial epithelial cells. Furthermore, we found that inhibition of endogenous AP-2beta expression by AP-2beta gene-specific small interfering RNAs effectively attenuated hTERT promoter-driven GFP expression, suppressed telomerase activity, accelerated telomere shortening, and inhibited tumor cell growth by induction of apoptosis in lung cancer cells. Our results demonstrate the tumor-specific activation of the hTERT promoter by AP-2beta and imply the potential of AP-2beta as a novel tumor marker or a cancer therapeutic target.

Interferon-gamma suppresses cyclooxygenase-2 promoter activity by inhibiting C-Jun and C/EBPbeta binding.

OBJECTIVE: Cyclooxygenase-2 (COX-2) and interferon gamma (IFNgamma) are overexpressed in vascular inflammatory and atherosclerotic lesions. We postulated that IFNgamma suppresses COX-2 expression at the transcriptional level. METHODS AND RESULTS: The effect of IFNgamma on COX-2 expression was evaluated in several types of human cells stimulated with phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1beta, or tumor necrosis factor (TNF) alpha. IFNgamma concentration-dependently inhibited COX-2 proteins and promoter activities induced by PMA or cytokines in human fibroblasts and monocytic and endothelial cells. PMA and cytokines stimulate binding of C-Jun, C-Fos, CCAAT/enhancer binding protein beta (C/EBPbeta), or NF-kappaB to their respective regulatory elements on COX-2 promoter. IFNgamma blocked C-Jun and C/EBPbeta but not C-Fos or p50 NF-kappaB binding as determined by in vitro binding assays and chromatin immunoprecipitation assay. p300 binding to COX-2 promoter was inhibited by IFNgamma in a manner comparable to C-Jun and C/EBPbeta binding. CONCLUSIONS: IFNgamma suppresses proinflammatory mediator-induced COX-2 transcription by selective inhibition of C-Jun and C/EBPbeta DNA binding activity and p300 recruitment in human cells. Because IFNgamma is coexpressed with COX-2 in vascular lesions, it may play a role in controlling COX-2-mediated inflammatory changes.

Induction of apoptosis by tumor suppressor FHIT via death receptor signaling pathway in human lung cancer cells.

FHIT is a novel tumor suppressor gene located at human chromosome 3p14.2. Restoration of wild-type FHIT in 3p14.2-deficient human lung cancer cells inhibits cell growth and induces apoptosis. In this study, we analyzed potential upstream/downstream molecular targets of the FHIT protein and found that FHIT specifically targeted and regulated death receptor (DR) genes in human non-small-cell lung cancer (NSCLC) cells. Exogenous expression of FHIT by a recombinant adenoviral vector (Ad)-mediated gene transfer upregulated expression of DR genes. Treatment with a recombinant TRAIL protein, a DR-specific ligand, in Ad-FHIT-transduced NSCLC cells considerably enhanced FHIT-induced apoptosis, further demonstrating the involvement of DRs in FHIT-induced apoptosis. Moreover, we also found that FHIT targeted downstream of the DR-mediated signaling pathway. FHIT overexpression disrupted mitochondrial membrane integrity and activated multiple pro-apoptotic proteins in NSCLC cell. These results suggest that FHIT induces apoptosis through a sequential activation of DR-mediated pro-apoptotic signaling pathways in human NSCLC cells.

Synergistic tumor suppression by coexpression of FUS1 and p53 is associated with down-regulation of murine double minute-2 and activation of the apoptotic protease-activating factor 1-dependent apoptotic pathway in human non-small cell lung cancer cells.

FUS1 is a novel tumor suppressor gene identified in human chromosome 3p21.3 region. Loss of expression and deficiency of posttranslational modification of FUS1 protein have been found in a majority of human lung cancers. Restoration of wild-type FUS1 in 3p21.3-deficient human lung cancer cells exhibited a potent tumor suppression function in vitro and in vivo. In this study, we evaluated the combined effects of FUS1 and tumor suppressor p53 on antitumor activity and explored the molecular mechanisms of their mutual actions in human non-small cell lung cancer (NSCLC) cells. We found that coexpression of FUS1 and p53 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly and synergistically inhibited NSCLC cell growth and induced apoptosis in vitro. We also found that a systemic treatment with a combination of FUS1 and p53 nanoparticles synergistically suppressed the development and growth of tumors in a human H322 lung cancer orthotopic mouse model. Furthermore, we showed that the observed synergistic tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression, the accumulation and stabilization of p53 protein, as well as the activation of the apoptotic protease-activating factor 1 (Apaf-1)-dependent apoptotic pathway in human NSCLC cells. Our results therefore provide new insights into the molecular mechanism of FUS1-mediated tumor suppression activity and imply that a molecular therapy combining two or more functionally synergistic tumor suppressors may constitute a novel and effective strategy for cancer treatment.

The 3p21.3 tumor suppressor NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells.

NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

Essential role of C-Rel in nitric-oxide synthase-2 transcriptional activation: time-dependent control by salicylate.

To determine the role of C-Rel in nitric-oxide synthase-2 (NOS-2) transcriptional activation, we evaluated the effect of lipopolysaccharide and interferon-gamma (LPS/IFNgamma) on C-Rel DNA binding in RAW 264.7. LPS/IFNgamma-stimulated C-Rel binding peaked at 4 to 8 h and declined at 24 h. Transfection of cells with a C-Rel small interfering RNA abrogated C-Rel binding at all time points. LPS/IFNgamma produced superoxide at 4 h, which subsided at 8 h. C-Rel binding and NOS-2 expression were abrogated by superoxide dismutase or apocynin at 4 h, suggesting a key role that superoxide plays in mediating C-Rel binding and NOS-2 transactivation only at 4 h. We have reported previously that salicylate at 10(-5) M inhibited LPS/IFNgamma-induced CCAAT/enhancer binding protein beta (C/EBPbeta) binding at 4 h but not at 8 or 24 h. A single dose of salicylate did not inhibit C-Rel binding at any time point. The addition of a second dose of salicylate 4 h before an indicated endpoint suppressed C-Rel but not C/EBPbeta or interferon-gamma-regulated factor-1 binding at 8 and 24 h. A single dose of salicylate added with LPS/IFNgamma inhibited NOS-2 expression only at 4 h. However, salicylate supplement inhibited NOS-2 promoter activities and mRNA and protein levels throughout 24 h. Signal profiling with a panel of inhibitors revealed time-dependent switch of signaling pathways. These results demonstrate temporal regulation of transactivator binding by LPS/IFNgamma via evolving signaling pathways. We propose that salicylate inhibits C/EBPbeta binding at 4 h and C-Rel binding at 8 and 24 h by targeting related kinases.

Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding.

Melatonin has been shown to be produced by nonpineal cells and possess anti-inflammatory actions in animal models. In the present study, we tested the hypothesis that melatonin suppresses the expression of proinflammatory genes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (INOS) by a common transcriptional mechanism. Melatonin but not tryptophan or serotonin inhibited lipopolysaccharide (LPS)-induced COX-2 and iNOS protein levels and promoter activities in RAW 264.7 cells in a time- and concentration-dependent manner. LPS or LPS plus interferon-gamma (IFNgamma) increased binding of all 5 isoforms of NF-kappaB to COX-2 and iNOS promoters. Melatonin selectively inhibited p52 binding without affecting p100 expression, p52 generation from p100, or p52 nuclear translocation. p52 acetylation was enhanced by LPS, which was abrogated by melatonin. Melatonin inhibited p300 histone acetyltransferase (HAT) activity and abrogated p300-augmented COX-2 and iNOS expression. HAT inhibitors suppressed LPS-induced p52 binding and acetylation to an extent similar to melatonin, and melatonin did not potentiate the effect of HAT inhibitors. These results suggest that melatonin inhibits COX-2 and iNOS transcriptional activation by inhibiting p300 HAT activity, thereby suppressing p52 acetylation, binding, and transactivation.

Role of p300 and PCAF in regulating cyclooxygenase-2 promoter activation by inflammatory mediators.

Coactivators p300 and CREB (cyclic adenosine monophosphate [cAMP]-response element binding protein)-binding protein (CBP) serve as an integrator for gene transcription. Their relative involvement in regulating cyclooxygenase-2 (COX-2) promoter activity had not been characterized. Using fibroblast and macrophage COX-2 transcription as a model, we determined p300 and CBP levels in nuclear extracts and their binding to a COX-2 promoter probe. CBP level was barely detectable and there was little CBP binding. In contrast, p300 was detectable in nucleus and its binding to a COX-2 promoter probe was enhanced by phorbol 12-myristate 13-acetate (PMA), interleukin-1 beta(IL-1 beta), or lipopolysaccharide (LPS). Binding of p300/CBP-associated factor (PCAF) was also up-regulated. COX-2 proteins and promoter activities induced by these agonists were augmented by p300 overexpression. Early region 1A (E1A), but not its deletion mutant, abrogated COX-2 expression induced by inflammatory mediators and with or without p300 overexpression. Molecular analysis of p300 revealed the requirement of multiple domains, including histone acetyltransferase (HAT) for COX-2 transactivation. Furthermore, roscovitine, an indirect inhibitor of p300 HAT, and histone deacetylase-1 transfection completely abolished COX-2 promoter activity. We conclude that p300 is the predominant coactivator that is essential for COX-2 transcriptional activation by proinflammatory mediators.