SHMT2 Induces Stemness and Progression of Head and Neck Cancer.

Various enzymes in the one-carbon metabolic pathway are closely related to the development of tumors, and they can all be potential targets for cancer therapy. Serine hydroxymethyltransferase2 (SHMT2), a key metabolic enzyme, is very important for the proliferation and growth of cancer cells. However, the function and mechanism of SHMT2 in head and neck cancer (HNC) are not clear. An analysis of The Cancer Genome Atlas (TCGA) data showed that the expression of SHMT2 was higher in tumor tissue than in normal tissue, and its expression was significantly associated with male sex, aggressive histological grade, lymph node metastasis, distant metastasis, advanced TNM stage, and lymphovascular invasion in HNC. SHMT2 knockdown in FADU and SNU1041 cell lines significantly inhibited cell proliferation, colony formation, migration, and invasion. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using TCGA data revealed that SHMT2 was closely related to cancer stem cell regulation and maintenance. Furthermore, we found that silencing SHMT2 inhibited the expression of stemness markers and tumor spheroid formation compared with a control group. On the contrary, stemness markers were significantly increased after SHMT2 overexpression in HEP-2 cells. Interestingly, we found that knocking down SHMT2 reduced the expression of genes related to the Notch and Wnt pathways. Finally, silencing SHMT2 significantly reduced tumor growth and decreased stemness markers in a xenograft model. Taken together, our study suggests that targeting SHMT2 may play an important role in inhibiting HNC progression.

Transcriptional Regulation of GDF15 by EGR1 Promotes Head and Neck Cancer Progression through a Positive Feedback Loop.

Growth and differentiation factor 15 (GDF15), a divergent member of the transforming growth factor-ß (TGF-ß) superfamily, has been reported to be overexpressed in different kinds of cancer types. However, the function and mechanism of GDF15 in head and neck cancer (HNC) remains unclear. The Cancer Genome Atlas (TCGA) data show that the expression of GDF15 is significantly associated with tumor AJCC stage, lymph vascular invasion and tumor grade in HNC. In this study, we confirmed that knockdown of GDF15 attenuated: cell proliferation, migration and invasion via regulation of EMT through a canonical pathway; SMAD2/3 and noncanonical pathways; PI3K/AKT and MEK/ERK in HNC cell lines. Furthermore, we found that early growth response 1 (EGR1) was a transcription factor of GDF15. Interestingly, we also demonstrated that GDF15 could regulate the expression of EGR1, which meant a positive feedback loop occurred between these two factors. Moreover, combined inhibition of both GDF15 and EGR1 in a HNC mouse xenograft model showed significantly decreased tumor volume compared to inhibition of EGR1 or GDF15 alone. Our study showed that the GDF15-EGR1 signaling axis may be a good target in HNC patients.

Defective Ca(2+) binding in a conserved binding site causes incomplete N-glycan processing and endoplasmic reticulum trapping of discoidin domain receptors.

An X-ray crystallographic study has suggested that vertebrate discoidin domain receptors (DDRs) have a conserved Ca(2+) binding site. DDR1 and DDR2 transfected in HEK293 cells were expressed mainly as 120 and 130 kDa forms, respectively, as they are sufficiently N-glycosylated. However, both of them showed the molecular weight of 110 kDa predominantly in the cells cultured with Ca(2+)-depleted media. DDR2-carrying D234A mutation at the conserved Ca(2+)-binding site expressed the 110 kDa form dominantly even in normal culture condition. DDR2 becomes 100 kDa form in glucose-depleted culture condition and its molecular weight increases up to 130 kDa with re-feeding glucose. However, in the mutant DDR2, the increase came to a halt at 110 kDa. The 110 kDa form had premature N-glycosyl carbohydrates and located predominantly within the endoplasmic reticulum. These results suggest that DDRs require Ca(2+)-binding to complete their N-glycan processing and generate the form targeted to cell membrane.

Single nucleotide polymorphisms in DKK3 gene are associated with prostate cancer risk and progression.

We had investigated whether sequence variants within DKK3 gene are associated with the development of prostate cancer in a Korean study cohort. We evaluated the association between 53 single nucleotide polymorphisms (SNPs) in the DKK3 gene and prostate cancer risk as well as clinical characteristics (PSA, clinical stage, pathological stage and Gleason score) in Korean men (272 prostate cancer subjects and 173 benign prostate hyperplasia subjects) using unconditional logistic regression analysis. Of the 53 SNPs and 25 common haplotypes, 5 SNPs and 4 haplotypes were associated with prostate cancer risk (P=0.02-0.04); 3 SNPs and 2 haplotypes were significantly associated with susceptibility to prostate cancer, however 2 SNPs and 2 haplotypes exhibited a significant protective effect on prostate cancer. Logistic analyses of the DKK3 gene polymorphisms with several prostate cancer related factors showed that several SNPs were significant; three SNPs and two haplotypes to PSA level, three SNPs and two haplotypes to clinical stage, nine SNPs and two haplotype to pathological stage, one SNP and one haplotypes to Gleason score. To the author's knowledge, this is the first report documenting that DKK3 polymorphisms are not only associated with prostate cancer but also related to prostate cancer-related factors.

Single nucleotide polymorphisms in fibroblast growth factor 23 gene, FGF23, are associated with prostate cancer risk.

OBJECTIVE: To determine whether sequence variants within the FGF23 gene are associated with the risk of developing prostate cancer in a Korean population. PATIENTS AND METHODS: Five common single nucleotide polymorphisms (SNPs) in the FGF23 gene were assessed in 272 patients with prostate cancer and 173 control subjects with benign prostatic hyperplasia. Single-locus analyses were conducted using conditional logistic regression. In addition, we performed a haplotype analysis for the five FGF23 SNPs tested. RESULTS: Three SNPs in the FGF23 gene (rs11063118, rs13312789 and rs7955866) were associated with an increased risk of prostate cancer in our study population. Odds ratios for homozygous variants vs wild-type variants ranged from 1.68 (95% confidence interval [CI]: 1.15-2.46) to 1.79 (95% CI: 1.16-2.75). CONCLUSION: This is the first study showing that genetic variations in FGF23 increase prostate cancer susceptibility.

The association between KL polymorphism and prostate cancer risk in Korean patients.

The Klotho (KL) gene is a classical "aging suppressor" gene. Although recent studies have shown that KL participates in the progression of several types of human cancers, the relationship between KL polymorphism and prostate cancer was unknown. The present study aimed to investigate the association between KL genetic polymorphisms and prostate cancer. Twenty-five common single nucleotide polymorphisms (SNPs) in KL gene (including KL gene polymorphism C1818T in exon 4) were assessed in 272 prostate cancer cases and 173 controls. Single-locus analyses were conducted using unconditional logistic regression. In addition, we did a haplotype analysis for the 25 KL SNPs tested. CC genotype of C1548T KL polymorphism had approximately twofold increased prostate cancer risk compared with the homozygous genotype TT and heterozygote CT (odds ratio 1.85 [95% CI, 1.09-3.12], P = 0.02). We also found that non-T allele carriers had significantly higher prostate cancer risk associated with the prostate cancer clinical characteristics (tumor stage or Gleason score). Our findings suggested that the C1548T polymorphism of KL gene is associated with the prostate cancer and may act as a risk factor for the development of prostate cancer.

Hypopigmentary effects of ethyl P-methoxycinnamate isolated from Kaempferia galanga.

We isolated crystals from the chloroform fraction of an ethanol extract of Kaempferia galanga and identified it as ethyl p-methoxycinnamate through nuclear magnetic resonance analysis. In the present study, we found that ethyl p-methoxycinnamate significantly decreased melanin synthesis in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH). In a cell-free system, however, ethyl p-methoxycinnamate did not directly inhibit tyrosinase, the rate-limiting enzyme of melanogenesis. Instead, it inhibited tyrosinase activity in B16F10 cells in a dose-dependent manner. Furthermore, Western blot analysis showed that ethyl p-methoxycinnamate decreased microphthalmia-associated transcription factor and tyrosinase levels in α-MSH-stimulated B16F10 cells. These results indicate that the pigment-inhibitory effect of ethyl p-methoxycinnamate results from downregulation of tyrosinase. Ethyl p-methoxycinnamate isolated from K. galanga could be developed as a skin whitening agent to treat hyperpigmentary disorders.

A circulating microRNA panel as a novel dynamic monitor for oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) has high recurrence and mortality rates despite advances in diagnosis and treatment. Therefore, it is necessary to identify new biomarkers for early detection, efficient monitoring, and prognosis prediction. Since microRNA (miRNA) is stable and detectable in serum, it has been reported to inform the diagnosis and monitor disease progression through liquid biopsy. In this study, a circulating specific miRNA panel in OSCC patients was developed, and its usefulness as a dynamic monitor was validated. Small RNAs were extracted from the serum of OSCC patients (n = 4) and normal controls (n = 6) and profiled using next-generation sequencing. NGS identified 42 differentially expressed miRNAs (DEmiRNAs) in serum between patients with OSCC and healthy controls, with threefold differences (p < 0.05). Combining the 42 DEmiRNAs and The Cancer Genome Atlas (TCGA) databases OSCC cohort, 9 overlapping DEmiRNAs were screened out. Finally, 4 significantly up-regulated miRNAs (miR-92a-3p, miR-92b-3p, miR-320c and miR-629-5p) were identified from OSCC patients via validation in the Chungnam National University Hospital cohort. Application of the specific miRNA panel for distinguishing OSCC patients from healthy controls produced specificity and sensitivity of 97.8 and 74%, respectively. In addition, the serum levels of these 4 miRNAs significantly decreased after complete surgical resection and increased after recurrence. We suggest that circulating 4-miRNA panel might be promising non-invasive predictors for diagnosing and monitoring the progression of patients with OSCC.

Mitochondrial Ribosomal Protein L14 Promotes Cell Growth and Invasion by Modulating Reactive Oxygen Species in Thyroid Cancer.

OBJECTIVES: The mitochondrial ribosomal protein L14 (MRPL14) is encoded by a nuclear gene and participates in mitochondrial protein translation. In this study, we aimed to investigate the role of MRPL14 in thyroid cancer. METHODS: We investigated the association between MRPL14 expression and clinicopathological features using The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) databases. Functional studies of MRPL14, including proliferation, migration, invasion, mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production, were performed in papillary thyroid cancer (PTC) cell lines (B-CPAP and KTC-1). RESULTS: Based on the TCGA dataset, PTC tissues lost mitochondrial integrity and showed dysregulated expression of overall mitoribosomal proteins (MRPs) compared with normal thyroid tissues. Of 78 MRPs, MRPL14 was highly expressed in thyroid cancer tissues. MRPL14 overexpression was significantly associated with advanced tumor stage, extrathyroidal extension, and lymph node metastasis. MRPL14 increased cell proliferation of thyroid cancer and promoted cell migration via epithelial-mesenchymal transition-related proteins. Moreover, MRPL14 knockdown reduced the expression of oxidative phosphorylation complex IV (MTCO1) and increased the accumulation of ROS. Cotreatment with a ROS scavenger restored cell proliferation and migration, which had been reduced by MRPL14 knockdown, implying that ROS functions as a key regulator of the oncogenic effects of MRPL14 in thyroid cancer cells. CONCLUSION: Our findings indicate that MRPL14 may promote cell growth, migration, and invasion by modulating ROS in thyroid cancer cells.

Sequence variants of Toll-like receptor 4 (TLR4) and the risk of prostate cancer in Korean men.

PURPOSE: Chronic inflammation has been considered a potential risk factor for prostate cancer. Toll-like receptors (TLRs) are important in the innate immune response to pathogens and in cross talk between innate immunity and adaptive immunity. In this study, sequence variants in the TLR4 gene were investigated to determine whether they were associated with prostate cancer risk in a Korean cohort. METHODS: An association study between 11 single-nucleotide polymorphisms (SNPs) of the TLR4 gene and prostate cancer was performed in 463 Korean male subjects including 240 prostate cancer patients and 223 healthy controls. SNPs were genotyped using the TaqMan assay, and their association with the risk of prostate cancer was evaluated using logistic regression analysis. RESULTS: The statistical analysis revealed that one SNP at the 3'UTR (rs11536889) showed significant association with the risk of prostate cancer (P (corr) = 0.005, OR = 1.81). One common haplotype (ht2) was also significantly associated with the risk of prostate cancer (P (corr) = 0.009, OR = 1.77). However, further analysis showed no association between any of the SNPs and prostate cancer prognostic factors such as the Gleason score or tumor stage. CONCLUSIONS: The findings of this study suggest that polymorphisms of the TLR4 gene might be associated with the risk of prostate cancer in Korean men.

Claudin-1 mediates progression by regulating EMT through AMPK/TGF-ß signaling in head and neck squamous cell carcinoma.

Claudin-1 (CLDN1), a major component of tight junction complexes in the epithelium, maintains cellular polarity, and plays a critical role in cell-to-cell communication as well as epithelial cell homeostasis. Although the role of CLDN1 has been widely studied in cancer, its role in the progression and the exact regulatory mechanisms, remain controversial. Using next-generation sequencing, we first analyzed the expression profiles of tumor/non-tumor paired tissue in patients with head and neck squamous cell carcinoma (HNSC) from public and local cohorts and found out that CLDN1 is upregulated in tumors compared to normal tissues. Next, its correlation with lymph node metastasis and poor prognosis was validated in the retrospective cohort, which collectively suggests CLDN1 as an oncogene in HNSC. As expected, the knockdown of CLDN1 inhibited invasive phenotypes by downregulating epithelial-to-mesenchymal transition (EMT) in vitro. To ascertain the regulatory mechanism of CLDN1 in HNSC analysis of GO term enrichment, KEGG pathways, and curated gene sets were used. As a result, CLDN1 was negatively associated with AMP-activated protein kinase (AMPK) and positively associated with transforming growth factor-ß (TGF-ß) signaling. In vitro mechanistic assay showed that CLDN1 inhibited AMPK phosphorylation by regulating AMPK upstream phosphatases, which led to inhibition of Smad2 activity. Intriguingly, the invasive phenotype of cancer cells increased by CLDN1 overexpression was rescued by AMPK activation, indicating a role of the CLDN1/AMPK/TGF-ß/EMT cascade in HNSC. Consistently in vivo, CLDN1 suppression significantly inhibited the tumor growth, with elevated AMPK expression, suggesting the novel observation of oncogenic CLDN1-AMPK signaling in HNSC.

Expression of human ß-defensin-2 in the prostate.

OBJECTIVE: To investigate the expression and regulation of human ß-defensin-2 (HBD-2) in the prostate. PATIENTS AND METHODS: Normal human prostate epithelial cell line (RWPE-1), human prostate cancer cell lines (DU-145, PC-3), and paraffin-embedded prostate tissue from patients with benign prostatic hyperplasia (BPH) were analysed by RT-PCR and immunohistochemical staining. HBD-2 expression was also analysed by RT-PCR and ELISA in RWPE-1 cells treated with lipopolysaccharide (LPS). Nuclear factor-κB (NF-κB) activation was assessed by IκBα immunoblotting and electrophoretic mobility shift assay (EMSA). RESULTS: BPH tissue and all of the tested prostate cell lines other than PC-3 constitutively express HBD-2 mRNA. HBD-2 protein was strongly detected in prostate gland tissue surrounded by inflammatory cells including macrophages. Exposure to LPS induced HBD-2 upregulation and NF-κB activation, as assessed by IκBα phosphorylation and degradation in RWPE-1 cells. Bay11-7082, an NF-κB inhibitor prevented LPS-induced HBD-2 production in RWPE-1 cells. CONCLUSIONS: Prostate epithelial cells may constitutively express HBD-2, and its expression was upregulated by LPS. Our data indicate that HBD-2 may be an important immunomodulatory factor in prostate function. Expression of HBD-2 in normal prostates and the potential role of HBD-2 in prostatitis and BPH should be addressed in the future.

Expression of resistin in the prostate and its stimulatory effect on prostate cancer cell proliferation.

OBJECTIVES: To determine whether resistin, a novel adipokine, induces prostate cancer cell proliferation. To identify the mechanisms underlying the activation of prostate cancer cells by resistin. MATERIALS AND METHODS: Semi-quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to investigate the intensity of prostate epithelial resistin expression. Human full-length resistin gene (RETN) was transfected into the PC-3 cells using the pEGFP-N1 vector to assess the effect of overexpression of resistin in prostate cancer cell line PC-3. Various concentrations of human recombinant protein resistin were added to the hormone-insensitive prostate cancer cell lines PC-3 and DU-145 for 48 h, and cell proliferation was assessed by a water-soluble tetrazolium salt assay. RESULTS: Human prostate cancer cell lines PC-3 and DU-145 were found to express the human resistin mRNA. Resistin protein was strongly detected in high-grade prostate cancer tissue, whereas BPH or low-grade prostate cancer tissue revealed fainter expression of resistin. Cell proliferation was stimulated by both the full-length resistin gene overexpression and resistin treatment. Akt phosphorylation occurred after addition of resistin to PC-3 and DU-145 cells. LY294002, a pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K), significantly inhibited PC-3 and DU-145 cell proliferation after resistin treatment. CONCLUSIONS: Resistin is expressed in human prostate cancers. Resistin induces prostate cancer cell proliferation through PI3K/Akt signalling pathways. The proliferative effect of resistin on prostate cancer cells may account in part for prostate cancer progression.

The effect of Curcumin on multi-level immune checkpoint blockade and T cell dysfunction in head and neck cancer.

BACKGROUND: Despite recent advances in understanding the complex immunologic dysfunction in the tumor microenvironment (TME), fewer than 20% of patients with head and neck squamous cell carcinoma (HNSCC) respond to immune checkpoint blockade (ICB). Thus, it is important to understand how inhibitory IC receptors maintain the suppressed dysfunctional TME, and to develop more effective combination immunotherapy. This study evaluated the immune-modulating effects of Curcumin, which has well-established anti-cancer and chemopreventive properties, and its long-term safety as a phytochemical drug. METHODS: We carried out the western blot and small interfering RNA (siRNA) transfection assay to evaluate the effects of Curcumin on IC ligands and IC ligands function in HNSCC. Through T-cell cytotoxicity assay and measurements of cytokine secretion, we assessed the effects of combination of Curcumin with programmed death-ligand 1 (PD-L1) Ab on cancer cell killing. Flow cytometry were used to analyze the effects of Curcumin on the expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain3 (TIM-3) on CD4, CD8 and Treg. Immunofluorescence, immunohistochemistry and western blot were used to detecte the cytokine (IFN-γ, Granzyme B), IC receptors (PD-1 and TIM-3) and its ligands (PD-L1, PD-L2, Galectin-9) in xenograft mouse model and 4-nitroquinoline-1-oxide (4-NQO) oral cancer model. RESULTS: We found that Curcumin decreased the expression of IC ligands such as PD-L1, PD-L2, and Galectin-9 in HNSCC, leading to regulation of epithelial-to-mesenchymal transition-associated tumor invasion. Curcumin also effectively restored the ability of CD8+ cytotoxic T cells to lyse cancer cells. To evaluate the effect of Curcumin on the TME further, the 4-NQO oral cancer model was used. Curcumin increased T-cell proliferation, tumor-infiltrating lymphocytes (TILs), and effector cytokines, and decreased the expression of PD-1, TIM-3, suppressive IC receptors and their ligands (PD-L1, PD-L2, and Galectin-9) in the TME, implying reinvigoration of the exhausted CD8+ T cells. In addition, Curcumin inhibited expression of CD4+CD25+FoxP3+ Treg cells as well as PD-1 and TIM-3. CONCLUSIONS: These results show that Curcumin reinvigorates defective T cells via multiple (PD-1 and TIM-3) and multi-level (IC receptors and its ligands) IC axis suppression, thus providing a rationale to combine Curcumin with conventional targeted therapy or ICB as a multi-faceted approach for treating patients with HNSCC.

EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma.

(1) Background: Nonthermal plasma (NTP) induces cell death in various types of cancer cells, providing a promising alternative treatment strategy. Although recent studies have identified new mechanisms of NTP in several cancers, the molecular mechanisms underlying its therapeutic effect on thyroid cancer (THCA) have not been elucidated. (2) Methods: To investigate the mechanism of NTP-induced cell death, THCA cell lines were treated with NTP-activated medium -(NTPAM), and gene expression profiles were evaluated using RNA sequencing. (3) Results: NTPAM upregulated the gene expression of early growth response 1 (EGR1). NTPAM-induced THCA cell death was enhanced by EGR1 overexpression, whereas EGR1 small interfering RNA had the opposite effect. NTPAM-derived reactive oxygen species (ROS) affected EGR1 expression and apoptotic cell death in THCA. NTPAM also induced the gene expression of growth arrest and regulation of DNA damage-inducible 45α (GADD45A) gene, and EGR1 regulated GADD45A through direct binding to its promoter. In xenograft in vivo tumor models, NTPAM inhibited tumor progression of THCA by increasing EGR1 levels. (4) Conclusions: Our findings suggest that NTPAM induces apoptotic cell death in THCA through a novel mechanism by which NTPAM-induced ROS activates EGR1/GADD45α signaling. Furthermore, our data provide evidence that the regulation of the EGR1/GADD45α axis can be a novel strategy for the treatment of THCA.

Head and Neck Cancer Cell Death due to Mitochondrial Damage Induced by Reactive Oxygen Species from Nonthermal Plasma-Activated Media: Based on Transcriptomic Analysis.

Mitochondrial targeted therapy is a next-generation therapeutic approach for cancer that is refractory to conventional treatments. Mitochondrial damage caused by the excessive accumulation of reactive oxygen species (ROS) is a principle of mitochondrial targeted therapy. ROS in nonthermal plasma-activated media (NTPAM) are known to mediate anticancer effects in various cancers including head and neck cancer (HNC). However, the signaling mechanism of HNC cell death via NTPAM-induced ROS has not been fully elucidated. This study evaluated the anticancer effects of NTPAM in HNC and investigated the mechanism using transcriptomic analysis. The viability of HNC cells decreased after NTPAM treatment due to enhanced apoptosis. A human fibroblast cell line and three HNC cell lines were profiled by RNA sequencing. In total, 1 610 differentially expressed genes were identified. Pathway analysis showed that activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were upstream regulators. Mitochondrial damage was induced by NTPAM, which was associated with enhancements of mitochondrial ROS (mtROS) and ATF4/CHOP regulation. These results suggest that NTPAM induces HNC cell death through the upregulation of ATF4/CHOP activity by damaging mitochondria via excessive mtROS accumulation, similar to mitochondrial targeted therapy.

Ceramide produces apoptosis through induction of p27(kip1) by protein phosphatase 2A-dependent Akt dephosphorylation in PC-3 prostate cancer cells.

Ceramide induces cell cycle arrest and apoptotic cell death associated with increased levels of p27(kip1). The aim of this study was to examine the effects of ceramide on p27(kip1) protein levels as a measure of cell cycle arrest and apoptosis. Results showed that ceramide increased p27(kip1) protein levels through activation of protein phosphatase 2A (PP2A) in PC-3 prostate cancer cells. Treatment of cells with the PP2A inhibitor okadaic acid or with PP2A-Cα siRNA inhibited ceramide-induced enhanced p27(kip1) protein expression and Akt dephosphorylation, and prevented Skp2 downregulation. Overexpression of constitutively active Akt attenuated ceramide-induced Skp2 downregulation and p27(kip1) upregulation. In addition, ceramide stimulated binding of the PP2A catalytic subunit PP2A-Cαß to Akt as assessed by immunoprecipitation experiments, indicating that PP2A is involved in the induction of p27(kip1) via inhibition of Akt pathway. Finally, whether PP2A can regulate p27(kip1) expression independently of Akt pathway was determined. Knockdown of PP2A-Cα with siRNA reduced p27(kip1) levels in the presence of Akt inhibitor. These data reveal that PP2A is a regulator of ceramide-induced p27(kip1) expression via Akt-dependent and Akt-independent pathways.

Role of bacterial DNA in macrophage activation by group B streptococci.

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.

Ceramide induces p38 MAPK-dependent apoptosis and Bax translocation via inhibition of Akt in HL-60 cells.

Ceramide induces apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells. However, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. In this study, the activation of p38 mitogen-activated protein kinase (MAPK) was found to be critical for the induction of apoptosis and subcellular redistribution of Bax. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a dominant-negative p38 MAPK attenuated DNA fragmentation, caspase-3 activation, and Bax translocation in response to ceramide. Overexpression of Akt also led to suppression of Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. We also provide evidence for cross-talk between p38 MAPK and Akt pathways. Expression of myr-Akt or inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 had no effect on p38 MAPK activation by ceramide as assessed by phosphorylation, while inhibition of p38 MAPK by a pharmacological inhibitor or a dominant-negative p38 inhibited Akt dephosphorylation in response to ceramide, suggesting that ceramide-induced p38 MAPK activation negatively regulates the Akt pathway.

Potentiation of ceramide-induced apoptosis by p27kip1 overexpression.

The cyclin-dependent kinase inhibitor p27kip1 (p27) has been implicated in the regulation of cell cycle and apoptosis. Recently, we have demonstrated that ceramide induces apoptotic cell death associated with increase in the level of p27 in HL-60 cells. In the present study, we showed that overexpression of p27 increases ceramide-induced apoptotic cell death in HL-60 cells. Furthermore, overexpression of p27 accelerated DNA fragmentation, PARP cleavage and cytochrome c release induced by ceramide. In addition, ceramide induced Bax expression independent of p27. These findings indicate that enhanced effect on apoptosis by p27 is associated with mitochondrial signaling which involves cytochrome c release.