Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.
Apoptosis/*drug effects ; Caspase 3/metabolism ; Chalcones/metabolism/*pharmacology ; Chemoprevention ; Cytochromes c/biosynthesis/secretion ; Down-Regulation ; Gene Expression ; HL-60 Cells ; Humans ; Immunoblotting ; Leukemia, Myeloid/*enzymology/pathology ; Membrane Potential, Mitochondrial/drug effects ; Mitochondria/enzymology ; Ornithine Decarboxylase/antagonists & inhibitors/genetics/*metabolism ; Reactive Oxygen Species/analysis/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Inflammation alters the neural stem cell (NSC) lineage from neuronal to astrogliogenesis. However, the underlying mechanism is elusive. Autophagy contributes to the decline in adult hippocampal neurogenesis under E. coli lipopolysaccharide (LPS) stimulation. SRY-box transcription Factor 2 (SOX2) is critical for NSC self-renewal and proliferation. In this study, we investigated the role of SOX2 in induced autophagy and hippocampal adult neurogenesis under LPS stimulation. LPS (5 ng•100 g -1 •hour -1 for 7 days) was intraperitoneally infused into male Sprague–Dawley rats (8 weeks old) to induce mild systemic inflammation. Beclin 1 and autophagy protein 12 (Atg12) were significantly upregulated concurrent with decreased numbers of Ki67- and doublecortin (DCX)-positive cells in the dentate gyrus. Synchronically, the levels of phospho(p)-mTOR, the p-mTOR/mTOR ratio, p-P85s6k, and the p-P85s6k/P85s6k ratio were suppressed. In contrast, SOX2 expression was increased. The fluorescence micrographs indicated that the colocalization of Beclin 1 and SOX2 was increased in the subgranular zone (SGZ) of the dentate gyrus. Moreover, increased S100β-positive astrocytes were colocalized with SOX2 in the SGZ. Intracerebroventricular infusion of 3-methyladenine (an autophagy inhibitor) effectively prevented the increases in Beclin 1, Atg12, and SOX2. The SOX2 + -Beclin 1 + and SOX2 + -S100β + cells were reduced. The levels of p-mTOR and p-P85s6k were enhanced. Most importantly, the number of DCX-positive cells was preserved. Altogether, these data suggest that LPS induced autophagy to inactivate the mTOR/P85s6k pathway, resulting in a decline in neural differentiation. SOX2 was upregulated to facilitate the NSC lineage, while the autophagy milieu could switch the SOX2-induced NSC lineage from neurogenesis to astrogliogenesis.

OBJECTIVETo evaluate apoptotic effects of cisplatin and cordycepin as single agent or in combination with cytotoxicity in oral cancer cells.

METHODSThe influences of cisplatin (2.5 μg/mL) and/or cordycepin treatment (10 or 100 μmol/L) to human OC3 oral cancer cell line were investigated by morphological observation for cell death appearance, methylthiazoletetrazolium (MTT) assay for cell viability, flow cytometry assay for cell apoptosis, and Western blotting for apoptotic protein expressions.

RESULTSData demonstrated that co-administration of cisplatin (2.5 μg/mL) and cordycepin (10 or 100 μmol/L) resulted in the enhancement of OC3 cell apoptosis compared to cisplatin or cordycepin alone treatment (24 h), respectively (P <0.05). In flow cytometry assay, percentage of cells arrested at subG1 phase with co-treatment of cordycepin and cisplatin (30%) was significantly higher than cisplatin (5%) or cordycepin (12%) alone group (P <0.05), confirming a synergistically apoptotic effect of cordycepin and cisplatin. In cellular mechanism study, co-treatment of cordycepin and cisplatin induced more stress-activated protein kinase/Jun terminal kinase (JNK), the expressions of caspase-7, and the cleavage of poly ADP-ribose polymerase (PARP) as compared to cisplatin or cordycepin alone treatment (P <0.05).

CONCLUSIONCisplatin and cordycepin possess synergistically apoptotic effect through the activation of JNK/caspase-7/PARP pathway in human OC3 oral cancer cell line.

Apoptosis ; drug effects ; Caspase 7 ; metabolism ; Cell Count ; Cell Line, Tumor ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cisplatin ; pharmacology ; Deoxyadenosines ; pharmacology ; Drug Synergism ; G1 Phase ; drug effects ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mouth Neoplasms ; pathology ; Phosphorylation ; drug effects ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVE: Hypermethylation of human papillomavirus (HPV) and host genes has been reported in cervical cancer. However, the degree of methylation of different HPV types relative to the severity of the cervical lesions remains controversial. Studies of the degree of methylation associated with the host gene and the HPV genome to the severity of cervical lesions are rare. We examined the association of methylation status between host genes and late gene 1 (L1) regions of HPV16, 18, 52, and 58 in cervical brushings. METHODS: Cervical brushings from 147 HPV-infected patients were obtained. The samples comprised normal (n=28), cervical intraepithelial neoplasia (CIN) 1 (n=45), CIN2 (n=13), and CIN3/carcinoma in situ (n=61). The methylation status of HPV and host genes was measured using bisulfite pyrosequencing and quantitative methylation-specific polymerase chain reaction (PCR). RESULTS: The degree of methylation of L1 in HPV16, 18, and 52 was associated with the severity of the cervical lesion. In HPV52, C-phosphate-G (CpG) sites 6368m, 6405m, and 6443m showed significantly higher methylation in lesions ≥CIN3 (p=0.005, 0.003, and 0.026, respectively). Methylation of most HPV types except HPV52 (r<−0.1) was positively correlated with the degree of methylation of host genes including PAX1 and SOX1 (0.4≤r≤0.7). Combining HPV methylation with PAX1 methylation improved the clustering for ≥CIN2. CONCLUSION: Our study showed that the degree of L1 methylation of HPV16, 18, and 52 but not 58 is associated with the severity of cervical lesions. The association between HPV methylation and host gene methylation suggests different responses of host cellular epigenetic machinery to different HPV genotypes.
Cervical Intraepithelial Neoplasia* ; DNA Methylation ; Epigenomics ; Genome ; Genotype ; Human papillomavirus 16 ; Humans ; Methylation* ; Papillomaviridae ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms

OBJECTIVE: We hypothesized that DNA methylation of development-related genes may occur in endometrial cancer (EC)/ovarian cancer (OC) and may be detected in cervical scrapings. METHODS: We tested methylation status by quantitative methylation-specific polymerase chain reaction for 14 genes in DNA pools of endometrial and OC tissues. Tissues of EC/normal endometrium, OC/normal ovary, were verified in training set using cervical scrapings of 10 EC/10 OC patients and 10 controls, and further validated in the testing set using independent cervical scrapings in 30 EC/30 OC patients and 30 controls. We generated cutoff values of methylation index (M-index) from cervical scrapings to distinguish between cancer patients and control. Sensitivity/specificity of DNA methylation biomarkers in detecting EC and OC was calculated. RESULTS: Of 14 genes, 4 (PTGDR, HS3ST2, POU4F3, MAGI2) showed hypermethylation in EC and OC tissues, and were verified in training set. POU4F3 and MAGI2 exhibited hypermethylation in training set were validated in independent cases. The mean M-index of POU4F3 is 78.28 in EC and 20.36 in OC, which are higher than that in controls (6.59; p<0.001 and p=0.100, respectively), and that of MAGI2 is 246.0 in EC and 12.2 in OC, which is significantly higher that than in controls (2.85; p<0.001 and p=0.480, respectively). Sensitivity and specificity of POU4F3/MAGI2 were 83%–90% and 69%–75% for detection of EC, and 61% and 62%–69% for the detection of OC. CONCLUSION: The findings demonstrate the potential of EC/OC detection through testing for DNA methylation in cervical scrapings.
Biomarkers ; DNA Methylation ; DNA ; Endometrial Neoplasms ; Endometrium ; Female ; Humans ; Methylation ; Ovarian Neoplasms ; Ovary ; Polymerase Chain Reaction ; Sensitivity and Specificity

BACKGROUND: Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity. METHODS: The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosomespecific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation. RESULTS The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition.Moreover, EVs decreased the lipid accumulation rate during adipogenic induction.