Identifying the top predictors of student well-being across cultures using machine learning and conventional statistics.

Alongside academic learning, there is increasing recognition that educational systems must also cater to students' well-being. This study examines the key factors that predict adolescent students' subjective well-being, indexed by life satisfaction, positive affect, and negative affect. Data from 522,836 secondary school students from 71 countries/regions across eight different cultural contexts were analyzed. Underpinned by Bronfenbrenner's bioecological theory, both machine learning (i.e., light gradient-boosting machine) and conventional statistics (i.e., hierarchical linear modeling) were used to examine the roles of person, process, and context factors. Among the multiple predictors examined, school belonging and sense of meaning emerged as the common predictors of the various well-being dimensions. Different well-being dimensions also had distinct predictors. Life satisfaction was best predicted by a sense of meaning, school belonging, parental support, fear of failure, and GDP per capita. Positive affect was most strongly predicted by resilience, sense of meaning, school belonging, parental support, and GDP per capita. Negative affect was most strongly predicted by fear of failure, gender, being bullied, school belonging, and sense of meaning. There was a remarkable level of cross-cultural similarity in terms of the top predictors of well-being across the globe. Theoretical and practical implications are discussed.

Defined tumor antigen-specific T cells potentiate personalized TCR-T cell therapy and prediction of immunotherapy response.

Personalized immunotherapy targeting tumor-specific antigens (TSAs) could generate efficient and safe antitumor immune response without damaging normal tissues. Although neoantigen vaccines have shown therapeutic effect in clinic trials, precise prediction of neoantigens from tumor mutations is still challenging. The host antitumor immune response selects and activates T cells recognizing tumor antigens. Hence, T cells engineered with T-cell receptors (TCRs) from these naturally occurring tumor antigen-specific T (Tas) cells in a patient will target personal TSAs in his/her tumor. To establish such a personalized TCR-T cell therapy, we comprehensively characterized T cells in tumor and its adjacent tissues by single-cell mRNA sequencing (scRNA-seq), TCR sequencing (TCR-seq) and in vitro neoantigen stimulation. Compared to bystander T cells circulating among tissues, Tas cells were characterized by tumor enrichment, tumor-specific clonal expansion and neoantigen specificity. We found that CXCL13 is a unique marker for both CD4+ and CD8+ Tas cells. Importantly, TCR-T cells expressing TCRs from Tas cells showed significant therapeutic effects on autologous patient-derived xenograft (PDX) tumors. Intratumoral Tas cell levels measured by CXCL13 expression precisely predicted the response to immune checkpoint blockade, indicating a critical role of Tas cells in the antitumor immunity. We further identified CD200 and ENTPD1 as surface markers for CD4+ and CD8+ Tas cells respectively, which enabled the isolation of Tas cells from tumor by Fluorescence Activating Cell Sorter (FACS) sorting. Overall, our results suggest that TCR-T cells engineered with Tas TCRs are a promising agent for personalized immunotherapy, and intratumoral Tas cell levels determine the response to immunotherapy.

DGKA interacts with SRC/FAK to promote the metastasis of non-small cell lung cancer.

Metastasis is responsible for the high mortality rate of lung cancer, but its underlying molecular mechanisms are poorly understood. Here, we demonstrated that the expression of diacylglycerol kinase alpha (DGKA) was elevated in the metastatic lesions of non-small cell lung cancer (NSCLC) and correlated with poor survival. Mechanistic studies revealed a direct physical interaction as well as a mutual regulation among DGKA, proto-oncogene tyrosine-protein kinase Src (SRC), and focal adhesion kinase 1 (FAK) proteins. The C-terminal domain of DGKA was responsible for the SRC SH3 domain binding, while the catalytic domain of DGKA interacted with the FREM domain of FAK. DGKA phosphorylated the SRC protein at Tyr416 and the FAK protein at Tyr397 to form and activate the DGKA/SRC/FAK complex, thus initiating the downstream WNT/ß-catenin and VEGF signaling pathways, promoting epithelial-mesenchymal transition (EMT) and angiogenesis, and resulting in the metastasis of NSCLC. DGKA knockdown inhibited the invasive phenotype of NSCLC cells in vitro. Pharmacologic ablation of DGKA inhibited the metastasis of NSCLC cells in vivo, and this was reversed by the overexpression of DGKA. These results suggested that DGKA was a potential prognostic biomarker as well as a promising therapeutic target for NSCLC, especially when there was lymphatic or distant metastasis.

Nucleoporin 93 mediates ß-catenin nuclear import to promote hepatocellular carcinoma progression and metastasis.

Nuclear pore complex (NPC) embedded in the nuclear envelope, is the only channel for macromolecule nucleocytoplasmic transportation and has important biological functions. However, the deregulation of specific nucleoporins (Nups) and NPC-Nup-based mechanisms and their function in tumour progression remain poorly understood. Here, we aimed to identify the Nups that contribute to HCC progression and metastasis in 729 primary hepatocellular carcinoma (HCC) cases using molecular, cytological, and biochemical techniques. Our results revealed elevated Nup93 expression in HCC tissues, especially in cases with metastasis, and was linked to worse prognosis. Furthermore, Nup93 knockdown suppressed HCC cell metastasis and proliferation, while Nup93 overexpression promoted these activities. We observed that Nup93 promotes HCC metastasis and proliferation by regulating ß-catenin translocation. In addition, we found that Nup93 interacted with ß-catenin directly, independent of importin. Furthermore, LEF1 and ß-catenin facilitated the Nup93-mediated metastasis and proliferation in HCC via a positive feedback loop. Thus, our findings provide novel insights into the mechanisms underlying the Nup93-induced promotion of HCC metastasis and suggest potential therapeutic targets in the LEF1-Nup93-ß-catenin pathway for HCC therapeutics.

DGKA Mediates Resistance to PD-1 Blockade.

Immunologic checkpoint blockade has been proven effective in a variety of malignancies. However, high rates of resistance have substantially hindered its clinical use. Understanding the underlying mechanisms may lead to new strategies for improving therapeutic efficacy. Although a number of signaling pathways have been shown to be associated with tumor cell-mediated resistance to immunotherapy, T cell-intrinsic resistant mechanisms remain elusive. Here, we demonstrated that diacylglycerol kinase alpha (Dgka) mediated T-cell dysfunction during anti-PD-1 therapy by exacerbating the exhaustion of reinvigorated tumor-specific T cells. Pharmacologic ablation of Dgka postponed T-cell exhaustion and delayed development of resistance to PD-1 blockade. Dgka inhibition also enhanced the efficacy of anti-PD-1 therapy. We further found that the expression of DGKA in cancer cells promoted tumor growth via the AKT signaling pathway, suggesting that DGKA might be a target in tumor cells as well. Together, these findings unveiled a molecular pathway mediating resistance to PD-1 blockade and provide a potential therapeutic strategy with combination immunotherapy.

Stress-glucocorticoid-TSC22D3 axis compromises therapy-induced antitumor immunity.

Psychological distress has long been suspected to influence cancer incidence and mortality. It remains largely unknown whether and how stress affects the efficacy of anticancer therapies. We observed that social defeat caused anxiety-like behaviors in mice and dampened therapeutic responses against carcinogen-induced neoplasias and transplantable tumors. Stress elevated plasma corticosterone and upregulated the expression of glucocorticoid-inducible factor Tsc22d3, which blocked type I interferon (IFN) responses in dendritic cell (DC) and IFN-γ+ T cell activation. Similarly, close correlations were discovered among plasma cortisol levels, TSC22D3 expression in circulating leukocytes and negative mood in patients with cancer. In murine models, exogenous glucocorticoid injection, or enforced expression of Tsc22d3 in DC was sufficient to abolish therapeutic control of tumors. Administration of a glucocorticoid receptor antagonist or DC-specific Tsc22d3 deletion reversed the negative impact of stress or glucocorticoid supplementation on therapeutic outcomes. Altogether, these results indicate that stress-induced glucocorticoid surge and Tsc22d3 upregulation can subvert therapy-induced anticancer immunosurveillance.

Diffuse large B cell lymphoma associated with chronic inflammation arising within atrial myxoma: aggressive histological features but indolent clinical behaviour.

AIMS: Primary cardiac lymphoma (PCL) is a rare neoplasm. PCL is fatal, unless it is diagnosed and treated early. Recently, a small number of cases of diffuse large B cell lymphoma (DLBCL) arising within atrial myxoma have been reported in immunocompetent patients and showed aggressive histological features but an indolent clinical behaviour. METHODS AND RESULTS: We present four unusual cases of Epstein-Barr virus (EBV)-positive DLBCL arising within atrial myxoma with detailed clinical, histological, immunophenotypical and genotypical features in immunocompetent patients, and review the literature for 11 similar cases. All the patients appeared to have morphological features of DLBCL, B lineage immunophenotype, high proliferative index and latency type III of EBV infection. They achieved complete tumour resection without chemotherapy or radiotherapy after surgery and were healthy at 3- and 7-month and 7- and 10-year follow-ups, respectively. CONCLUSIONS: We suggest that this lymphoma should be regarded as a unique DLBCL associated with chronic inflammation (DLBCL-CI) because of an indolent clinical behaviour to avoid excessive or unnecessary treatments. In addition, early accurate diagnosis and complete resection of this tumour are crucial for optimal patient outcome.

Melatonin inhibits AP-2ß/hTERT, NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto C signaling to enhance the antitumor activity of berberine in lung cancer cells.

Melatonin, a molecule produced throughout the animal and plant kingdoms, and berberine, a plant derived agent, both exhibit antitumor and multiple biological and pharmacological effects, but they have never been combined altogether for the inhibition of human lung cancers. In this study, we investigated the role and underlying mechanisms of melatonin in the regulation of antitumor activity of berberine in lung cancer cells. Treatment with melatonin effectively increased the berberine-mediated inhibitions of cell proliferation, colony formation and cell migration, thereby enhancing the sensitivities of lung cancer cells to berberine. Melatonin also markedly increased apoptosis induced by berberine. Further mechanism study showed that melatonin promoted the cleavage of caspse-9 and PARP, enhanced the inhibition of Bcl2, and triggered the releasing of cytochrome C (Cyto C), thereby increasing the berberine-induced apoptosis. Melatonin also enhanced the berberine-mediated inhibition of telomerase reverses transcriptase (hTERT) by down-regulating the expression of AP-2ß and its binding on hTERT promoter. Moreover, melatonin enhanced the berberine-mediated inhibition of cyclooxygenase 2 (COX-2) by inhibiting the nuclear translocation of NF-κB and its binding on COX-2 promoter. Melatonin also increased the berberine-mediated inhibition of the phosphorylated Akt and ERK. Collectively, our results demonstrated that melatonin enhanced the antitumor activity of berberine by activating caspase/Cyto C and inhibiting AP-2ß/hTERT, NF-κB/COX-2 and Akt/ERK signaling pathways. Our findings provide new insights in exploring the potential therapeutic strategies and novel targets for lung cancer treatment.

Lasiodin inhibits proliferation of human nasopharyngeal carcinoma cells by simultaneous modulation of the Apaf-1/caspase, AKT/MAPK and COX-2/NF-κB signaling pathways.

Rabdosia serra has been widely used for the treatment of the various human diseases. However, the antiproliferative effects and underlying mechanisms of the compounds in this herb remain largely unknown. In this study, an antiproliferative compound against human nasopharyngeal carcinoma (NPC) cells from Rabdosia serra was purified and identified as lasiodin (a diterpenoid). The treatment with lasiodin inhibited cell viability and migration. Lasiodin also mediated the cell morphology change and induced apoptosis in NPC cells. The treatment with lasiodin induced the Apaf-1 expression, triggered the cytochrome-C release, and stimulated the PARP, caspase-3 and caspase-9 cleavages, thereby activating the apoptotic pathways. The treatment with lasiodin also significantly inhibited the phosphorylations of the AKT, ERK1/2, p38 and JNK proteins. The pretreatment with the AKT or MAPK-selective inhibitors considerably blocked the lasiodin-mediated inhibition of cell proliferation. Moreover, the treatment with lasiodin inhibited the COX-2 expression, abrogated NF-κB binding to the COX-2 promoter, and promoted the NF-κB translocation from cell nuclei to cytosol. The pretreatment with a COX-2-selective inhibitor abrogated the lasiodin-induced inhibition of cell proliferation. These results indicated that lasiodin simultaneously activated the Apaf-1/caspase-dependent apoptotic pathways and suppressed the AKT/MAPK and COX-2/NF-κB signaling pathways. This study also suggested that lasiodin could be a promising natural compound for the prevention and treatment of NPC.

TFAP2B overexpression contributes to tumor growth and a poor prognosis of human lung adenocarcinoma through modulation of ERK and VEGF/PEDF signaling.

BACKGROUND: TFAP2B is a member of the AP2 transcription factor family, which orchestrates a variety of cell processes. However, the roles of TFAP2B in regulating carcinogenesis remain largely unknown. Here, we investigated the regulatory effects of TFAP2B on lung adenocarcinomas growth and identified the underlying mechanisms of actions in non-small cell lung cancer (NSCLC) cells. METHODS: We first examined the expression of TFAP2B in lung cancer cell lines and tumor tissues. We also analyzed the prognostic predicting value of TFAP2B in lung adenocarcinomas. Then we investigated the molecular mechanisms by which TFAP2B knockdown or overexpression regulated lung cancer cell growth, angiogenesis and apoptosis, and further confirmed the role of TFAP2B in tumor growth in a lung cancer xenograft mouse model. RESULTS: TFAP2B was highly expressed in NSCLC cell lines and tumor tissues. Strong TFAP2B expression showed a positive correlation with the poor prognoses of patients with lung adenocarcinomas (P < 0.001). TFAP2B knockdown by siRNA significantly inhibited cell growth and induced apoptosis in NSCLC cells in vitro and in a lung cancer subcutaneous xenograft model, whereas TFAP2B overexpression promoted cell growth. The observed regulation of cell growth was accompanied by the TFAP2B-mediated modulation of the ERK/p38, caspase/cytochrome-c and VEGF/PEDF-dependent signaling pathways in NSCLC cells. CONCLUSIONS: These results indicate that TFAP2B plays a critical role in regulating lung adenocarcinomas growth and could serve as a promising therapeutic target for lung cancer treatment.

CPSF4 activates telomerase reverse transcriptase and predicts poor prognosis in human lung adenocarcinomas.

The elevated expression and activation of human telomerase reverse transcriptase (hTERT) is associated with the unlimited proliferation of cancer cells. However, the excise mechanism of hTERT regulation during carcinogenesis is not well understood. In this study, we discovered cleavage and polyadenylation specific factor 4 (CPSF4) as a novel tumor-specific hTERT promoter-regulating protein in lung cancer cells and identified the roles of CPSF4 in regulating lung hTERT and lung cancer growth. The ectopic overexpression of CPSF4 upregulated the hTERT promoter-driven report gene expression and activated the endogenous hTERT mRNA and protein expression and the telomerase activity in lung cancer cells and normal lung cells. In contrast, the knockdown of CPSF4 by siRNA had the opposite effects. CPSF4 knockdown also significantly inhibited tumor cell growth in lung cancer cells in vitro and in a xenograft mouse model in vivo, and this inhibitory effect was partially mediated by decreasing the expression of hTERT. High expression of both CPSF4 and hTERT proteins were detected in lung adenocarcinoma cells by comparison with the normal lung cells. Tissue microarray immunohistochemical analysis of lung adenocarcinomas also revealed a strong positive correlation between the expression of CPSF4 and hTERT proteins. Moreover, Kaplan-Meier analysis showed that patients with high levels of CPSF4 and hTERT expression had a significantly shorter overall survival than those with low CPSF4 and hTERT expression levels. Collectively, these results demonstrate that CPSF4 plays a critical role in the regulation of hTERT expression and lung tumorigenesis and may be a new prognosis factor in lung adenocarcinomas.

TFAP2A regulates nasopharyngeal carcinoma growth and survival by targeting HIF-1α signaling pathway.

TFAP2A is a transcription factor that orchestrates a variety of cell processes, including cell growth and tissue differentiation. However, the regulation of TFAP2A in human nasopharyngeal carcinoma tumorigenesis and its precise mechanism of action remain largely unknown. In this study, we investigated the biologic role and clinical significance of TFAP2A in nasopharyngeal carcinoma growth and progression and identified the underlying molecular mechanisms. We found that TFAP2A was highly expressed in various nasopharyngeal carcinoma cell lines and tumor tissue specimens and was significantly correlated with hypoxia-inducible factor-1α (HIF-1α) expression. A positive correlation of TFAP2A overexpression with advanced tumor stage, local invasion, clinical progression, and poor prognosis of patients with nasopharyngeal carcinomas were also observed. Moreover, we found that knockdown of TFAP2A expression by siRNA significantly inhibited tumor cell growth in nasopharyngeal carcinoma cell lines and in a subcutaneous xenograft mouse model by targeting the HIF-1α-mediated VEGF/pigment epithelium-derived factor (PEDF) signaling pathway. Treatment of nasopharyngeal carcinoma cells with TFAP2A siRNA dramatically inhibited the expression and the release of VEGF protein but did not change the level of PEDF protein, resulting in a significant reduction of the ratio of VEGF/PEDF. Pretreatment with a HIF-1α siRNA did not significantly change the TFAP2A siRNA-mediated inhibition in cell viability. Our results indicate that TFAP2A regulates nasopharyngeal carcinoma growth and survival through the modulation of the HIF-1α-mediated VEGF/PEDF signaling pathway, and suggest that TFAP2A could be a potential prognostic biomarker and therapeutic target for nasopharyngeal carcinoma treatment.

Upregulation of cleavage and polyadenylation specific factor 4 in lung adenocarcinoma and its critical role for cancer cell survival and proliferation.

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3' ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.

Berberine Targets AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth.

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2ß expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2ß by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

Ursolic acid simultaneously targets multiple signaling pathways to suppress proliferation and induce apoptosis in colon cancer cells.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Melatonin potentiates the antiproliferative and pro-apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant-derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro-apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX-2, and p300/NF-κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX-2 expression, promoted p300 and NF-κB translocation from cell nuclei to cytoplasm, and abrogated NF-κB binding and p300 recruitment to COX-2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro-apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.

Nicotine promotes proliferation of human nasopharyngeal carcinoma cells by regulating α7AChR, ERK, HIF-1α and VEGF/PEDF signaling.

Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC) cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.

Melatonin suppresses proinflammatory mediators in lipopolysaccharide-stimulated CRL1999 cells via targeting MAPK, NF-κB, c/EBPß, and p300 signaling.

Melatonin is an indoleamine secreted by the pineal gland as well as a plant-derived product that exerts potential anti-inflammatory properties, but the mechanisms of action remain unclear. Here, we investigated the roles of melatonin in regulation of proinflammatory mediators and identified the underlying mechanisms in human vascular smooth muscle (VSM) cell line CRL1999 stimulated by lipopolysaccharide (LPS). We found that treatment with melatonin significantly inhibited the production and expression of TNF-α and interleukin (IL)-1ß, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, prostaglandin E(2) (PGE2), and nitric oxide (NO) in a dose-dependent manner. Moreover, we also found that the suppression of proinflammatory mediators by melatonin was mediated through inhibition of MAPK, NF-κB, c/EBPß, and p300 signaling in LPS-stimulated CRL1999 cells. Treatment with melatonin markedly inhibited phosphorylation of ERK1/2, JNK, p38 MAPK, IκB-α, and c/EBPß, blocked binding of NF-κB and c/EBPß to promoters, and suppressed p300 histone acetyltransferase (HAT) activity and p300 HAT-mediated NF-κB acetylation. Transfection with an ERK-, IκB-, or c/EBPß-specific siRNA or pretreatment with an ERK-, p38 MAPK-, or p300-selective inhibitor considerably abrogated the melatonin-mediated inhibition of proinflammatory mediators. Conversely, exogenous overexpression of a constitutively active p300, but not its HAT mutant, effectively reversed the melatonin-mediated inhibitions. Collectively, these results indicate that melatonin suppresses proinflammatory mediators by simultaneously targeting the multiple signaling such as ERK/p38 MAPK, c/EBPß, NF-κB, and p300, in LPS-stimulated VSM cell line CRL1999, and suggest that melatonin is a potential candidate compound for the treatment of proinflammatory disorders.

Simultaneous modulation of COX-2, p300, Akt, and Apaf-1 signaling by melatonin to inhibit proliferation and induce apoptosis in breast cancer cells.

Melatonin exhibits anti-inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA-MB-361 breast cancer cells. Melatonin at pharmacological concentrations (10(-3) m) significantly suppressed cell proliferation and induced apoptosis in a dose-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of COX-2, p300, and NF-κB signaling. Melatonin significantly inhibited COX-2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300-mediated NF-κB acetylation, thereby blocking NF-κB binding and p300 recruitment to COX-2 promoter. Pretreatment with a COX-2- or p300-selective inhibitor abrogated the melatonin-induced inhibition of cell proliferation, whereas PGE2 treatment or COX-2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK-3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K- or an Akt-selective inhibitor or an Akt-specific siRNA blocked the melatonin-mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf-1 expression, triggered cytochrome C release, and stimulated caspase-3 and caspase-9 activities and cleavage, leading to an activation of the Apaf-1-dependent apoptotic pathway. Pretreatment with an Apaf-1-specific siRNA effectively attenuated the melatonin-induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA-MB-361 breast cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-κB, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.