Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy.

BACKGROUND: Emerging studies showed curcumin can inhibit glioblastoma and breast cancer cells via regulating ferroptosis. However, the role of ferroptosis in the inhibitory effect of curcumin on non-small-cell lung cancer (NSCLC) remains unclear. METHODS: Cell counting kit-8 (CCK-8) assay was used to measure the viability of A549 and H1299 cells under different conditions. Cell proliferation was examined by Ki67 immunofluorescence. The morphological changes of cells and tumor tissues were observed by optical microscope and hematoxylin and eosin (H&E) staining. Intracellular reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and iron contents were determined by corresponding assay kit. The related protein expression levels were detected by western blot and immunohistochemistry. Transmission electron microscope was used to observe ultrastructure changes of A549 and H1299 cells. RESULTS: Curcumin inhibited tumor growth and cell proliferation, but promoted cell death. Characteristic changes of ferroptosis were observed in curcumin group, including iron overload, GSH depletion and lipid peroxidation. Meanwhile, the protein level of ACSL4 was higher and the levels of SLC7A11 and GPX4 were lower in curcumin group than that in control group. Incubation of ferroptosis inhibitors ferrostatin-1 (Fer-1) or knockdown of iron-responsive element-binding protein 2 (IREB2) notably weakened curcumin-induced anti-tumor effect and ferroptosis in A549 and H1299 cells. Further investigation suggested that curcumin induced mitochondrial membrane rupture and mitochondrial cristae decrease, increased autolysosome, increased the level of Beclin1 and LC3, and decreased the level of P62. Curcumin-induced autophagy and subsequent ferroptosis were both alleviated with autophagy inhibitor chloroquine (CQ) or siBeclin1. CONCLUSION: Curcumin induced ferroptosis via activating autophagy in NSCLC, which enhanced the therapeutic effect of NSCLC.

Intermittent hypoxia-induced autophagy via AMPK/mTOR signaling pathway attenuates endothelial apoptosis and dysfunction in vitro.

PURPOSE: The aim of this study was to examine whether or not intermittent hypoxia (IH) upregulated autophagy and the contributions of autophagy to endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs). METHOD: HUVECs were incubated under normoxia and IH conditions. After 3-, 6-, 12-, and 24-h exposure, the autophagic vacuoles and autophagosomes were observed by transmission electron microscopy and monodansylcadaverine staining. The protein levels of autophagy-related biomarkers and AMPK/mTOR pathway were measured by Western blot. The apoptosis-related proteins and the percentage of apoptotic cells were evaluated by Western blot and flow cytometry, respectively, while the levels of endothelial function biomarkers were assessed by ELISA. RESULTS: IH induced autophagy, as determined by the increased numbers of the autophagic vacuoles, autophagosomes, and by the elevated levels of Beclin-1 protein, the LC3II/LC3I ratio, and p62 degradation. IH-induced autophagic flux peaked at 12-h duration and weakened at 24 h. IH increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR, while compound C restored the alteration. A significant decrease in the Bcl-2 level and the Bcl-2/Bax ratio and a significant increase in the protein expression levels of Bax and cleaved caspase 3 and in the percentage of apoptosis were observed under IH exposure. Moreover, the NO level was reduced, while the ET-1 and VEGF levels were raised under IH condition. These alterations were suppressed by the pretreatment of 3-methyladenine. CONCLUSIONS: IH upregulates autophagy through AMPK/mTOR pathway in HUVECs in vitro, which might be protective against endothelial apoptosis and dysfunction caused by IH.

Influence of coexistence of mild OSA on airway mucus hypersecretion in patients with COPD.

The coexistence of chronic obstructive pulmonary disease (COPD) and obstructive sleep apnea (OSA) can cause multiple system damage, and the main physiological mechanisms are continuous hypoxia and intermittent hypoxia (IH). Airway mucus hypersecretion is an important clinical feature of COPD, which can cause a progressive decline of lung function, acute COPD aggravation, and disease progression. The purpose of our study is to determine the influence of the coexistence of mild OSA on airway mucus hypersecretion. Clinical data and airway epithelial samples of 36 subjects were collected. The average fluorescence intensity of MUC5AC and the number of goblet cells were measured through immunofluorescence staining. MUC5AC expression was measured in human bronchial epithelial (HBE) cells exposed to normoxia, IH, particulate matter (PM), and PM + IH using real-time quantitative polymerase chain reaction and western blotting. FEV1% pred and FEV1/FVC were higher in patients with COPD-OSA overlap syndrome (OS) than in patients with COPD alone. Patients with OS had less sputum volume than patients with COPD alone. MUC5AC expression and the number of goblet cells in the airway epithelium in the COPD alone group were significantly higher than those in the OS groups. The PM + IH group had lower MUC5AC mRNA and protein expression in HBE cells than the PM group. The coexistence of mild OSA may reduce goblet cell proliferation and MUC5AC expression in the airway epithelium of patients with COPD. Mild IH inhibited PM-induced up-regulation of MUC5AC expression in the mRNA and protein levels in HBE cells.

Osteoporosis in a Rat Model Co-Exposed to Cigarette Smoke and Intermittent Hypoxia.

Purpose: There are few studies on osteoporosis in chronic obstructive pulmonary disease-obstructive sleep apnea overlap syndrome, and the results obtained are inconsistent. The purpose of our study is to observe the occurrence of osteoporosis and its possible mechanism in rat model co-exposed to cigarette smoke and intermittent hypoxia. Materials and Methods: The rats were randomly divided into four groups: air exposed group, cigarette smoke (CS) exposed group, 10% concentration of intermittent hypoxia exposed group, CS combined with 10% concentration of intermittent hypoxia exposed group. All animals completed lung function and lung tissue morphology assessment. The femurs were examined by microcomputer tomography (microCT). Tartrate-resistant acidic phosphatase (TRAP) staining was used to evaluate the osteoclasts. We also assessed the interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in peripheral blood. Results: There was no difference in the femoral length between each group, but the quantitative analyses of microCT showed that compared with the air exposed group, the percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), cortical thickness (Ct.Th) and bone mineral density (BMD) decreased, and the trabecular separation (Tb.Sp) and the proportion of trap-positive cells increased significantly in the overlapping exposed group. There were higher levels of BV/TV in the overlapping group than CS exposed group. Compared with the intermittent hypoxia exposed group, there were lower levels of Tb.Th and Ct.Th and higher levels of Tb.Sp in the overlapping exposed group. However, there was no statistical difference of trap-positive cell between the overlapping exposed group and the CS exposed single group or the intermittent hypoxia exposed group. There were higher levels of IL-6 and TNF-α in the overlapping exposed group than those in the air-exposed group. Conclusion: Bone destruction increased in the overlapping exposed rat model compared with the rat exposed to air, which may be related to the upregulation of inflammation.

Heat Shock Protein 90α-Dependent B-Cell-2-Associated Transcription Factor 1 Promotes Hepatocellular Carcinoma Proliferation by Regulating MYC Proto-Oncogene c-MYC mRNA Stability.

B-cell lymphoma 2 (Bcl-2)-associated transcription factor 1 (Bclaf1) is known to be involved in diverse biological processes, but, to date, there has been no evidence for any functional role of Bclaf1 in hepatocellular carcinoma (HCC) progression. Here, we demonstrate that Bclaf1 is frequently up-regulated in HCC and that Bclaf1 up-regulation is associated with Edmondson grade, lower overall survival rates, and poor prognosis. Overexpression of Bclaf1 in HCC cell lines HepG2 and Huh7 promoted proliferation considerably, whereas Bclaf1 knockdown had the opposite effect. Xenograft tumors grown from Bclaf1 knockdown Huh7 cells had smaller tumor volumes than tumors grown from control cells. Furthermore, our study describes MYC proto-oncogene (c-Myc) as a downstream target of Bclaf1, given that Bclaf1 regulates c-MYC expression posttranscriptionally by its RS domain. To exert this function, Bclaf1 must interact with the molecular chaperone, heat shock protein 90 alpha (Hsp90α). In HCC tissue samples, Hsp90α levels were also increased significantly and Hsp90α-Bclaf1 interaction was enhanced. Bclaf1 interacts with the C-terminal domain of Hsp90α, and this interaction is disrupted by the C-terminal domain inhibitor, novobiocin (NB), resulting in proteasome-dependent degradation of Bclaf1. Moreover, NB-induced disruption of Hsp90α-Bclaf1 interaction dampened the production of mature c-MYC mRNA and attenuated tumor cell growth in vitro and in vivo. Conclusion: Our findings suggest that Bclaf1 affects HCC progression by manipulating c-MYC mRNA stability and that the Hsp90α/Bclaf1/c-Myc axis might be a potential target for therapeutic intervention in HCC.

Dental fear and its possible relationship with periodontal status in Chinese adults: a preliminary study.

BACKGROUND: The aim of the present study was to describe the characteristics of dental fear of Chinese adult patients with periodontal disease and provide information for clinical assessment. METHODS: A total of 1203 dental patients completed questionnaires that included Corach's Dental Anxiety Scales (DAS), Dental Fear Survey (DFS) and the short-form Dental Anxiety Inventory (S-DAI). Among all the patients, 366 cases were self-reported periodontal disease. The general characteristics were described, such as socio-demographics, dental attendances and oral health behaviors. The statistical analysis was performed by t-test, Mann-Whitney U test and linear regression respectively to evaluate correlations between dental fear and general characteristics according to the three scales. RESULTS: The prevalence of dental fear was 74% among 1203 patients, 23.4% of total with high dental fear, while 27.3% in the patients with periodontal disease. The average score of DAS and DFS for patients with periodontal disease was significantly higher than those without periodontal disease. The regression analysis indicated that gender, age, periodontal status, dental attendances and oral health behaviors were correlated with dental fear. Among 366 patients with periodontal disease, gender, dental attendances and oral health behaviors had correlation with dental fear. The analysis of DFS scale exhibited that 'drilling with handpiece' and 'injecting the anesthetic' were the most important factors to contribute to dental fear. CONCLUSIONS: There was high prevalence of dental fear in Chinese adult patients, particularly in patients with periodontal disease, and high level of dental fear may lead to poor periodontal status.

Knowledge, awareness, and behaviors of endocrinologists and dentists for the relationship between diabetes and periodontitis.

AIMS: This study aimed to compare the opinions of dentists and endocrinologists regarding diabetes mellitus (DM) and periodontitis, and to investigate the possible effects on their practice. METHODS: Cross-sectional data were collected from 297 endocrinologists and 134 dentists practicing in southern China using two separated questionnaires. Questions were close-ended or Likert-scaled. Statistical analyses were done by descriptive statistics, bivariate and binary logistic regression analysis. RESULTS: Compared with endocrinologists, dentists presented more favorable attitudes for the relationship of DM and periodontitis (P<0.001). 61.2% of dentists reported they would frequently refer patients with severe periodontitis for DM evaluation, while only 26.6% of endocrinologists reported they would frequently advise patients with DM to visit a dentist. Nearly all of the respondents (94.4%) agreed that the interdisciplinary collaboration should be strengthened. The logistic regression analysis exhibited that respondents with more favorable attitudes were more likely to advise a dental visit (P=0.003) or to screen for DM (P=0.006). CONCLUSIONS: Endocrinologists and dentists are not equally equipped with the knowledge about the relationship between DM and periodontitis, and there is a wide gap between their practice and the current evidence, especially for endocrinologists. It's urgent to take measures to develop the interdisciplinary education and collaboration among the health care providers.

Berberine inhibits norepinephrine-induced apoptosis in neonatal rat cardiomyocytes via inhibiting ROS-TNF-α-caspase signaling pathway.

OBJECTIVE: To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes. METHODS: The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined. RESULTS: NE at a concentration of 50 µ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 µ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 µ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h. CONCLUSION: The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Yohimbine enhances protection of berberine against LPS-induced mouse lethality through multiple mechanisms.

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS-induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-ß and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms.

AIM: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. METHODS: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50 mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. RESULTS: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. CONCLUSION: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

Sodium valproate induces mitochondria-dependent apoptosis in human hepatoblastoma cells.

BACKGROUND: Sodium valproate inhibits proliferation in neuroblastoma and glioma cells, and inhibits proliferation and induces apoptosis in hepatoblastoma cells. Information describing the molecular pathways of the antitumor effects of sodium valproate is limited; therefore, we explored the mechanisms of action of sodium valproate in the human hepatoblastoma cell line, HepG2. METHODS: The effects of sodium valproate on the proliferation of HepG2 cells were evaluated by the Walsh-schema transform and colony formation assays. Sodium valproate-induced apoptosis in HepG2 cells was investigated with fluorescence microscopy to detect morphological changes; by flow cytometry to calculate DNA ploidy and apoptotic cell percentages; with Western blotting analyses to determine c-Jun N-terminal kinases (JNK), p-JNK, Bcl-2, Bax, and caspase-3 and -9 protein expression levels; and using JC-1 fluorescence microscopy to detect the membrane potential of mitochondria. Statistical analyses were performed using one-way analysis of variance by SPSS 13.0 software. RESULTS: Our results indicated that sodium valproate treatment inhibited the proliferation of HepG2 cells in a dose-dependent manner. Sodium valproate induced apoptosis in HepG2 cells as it: caused morphologic changes associated with apoptosis, including condensed and fragmented chromatin; increased the percentage of hypodiploid cells in a dose-dependent manner; increased the percentage of annexin V-positive/propidium iodide-negative cells from 9.52% to 74.87%; decreased JNK and increased phosphate-JNK protein expression levels; reduced the membrane potential of mitochondria; decreased the ratio of Bcl-2/Bax; and activated caspases-3 and -9. CONCLUSION: Sodium valproate inhibited the proliferation of HepG2 cells, triggered mitochondria-dependent HepG2 cell apoptosis and activated JNK.

Effect of cleidocranial dysplasia-related novel mutation of RUNX2 on characteristics of dental pulp cells and tooth development.

Cleidocranial dysplasia (CCD) is an autosomal-dominant disorder caused by a lack of function of one or more alleles of the RUNX2 gene. Mutations of the RUNX2 gene were analyzed in a family with CCD, and a novel nonsense mutation was identified, c. 1096G > T, p.E366X, which was predicted to cause a number of potential dysfunctions. Western blot analysis showed that the novel mutation created a shortened protein product, which lost 155 aa in the C-terminal domain. The mutant protein was detected to be localized mostly in the cytoplasm, not in the nucleus, which demonstrated that transport of the RUNX2 protein into the nucleus was disturbed by the p.E366X mutation. For the first time, RUNX2(+/m) dental pulp cells (DPCs) were isolated from two permanent incisors of the CCD patient. Compared to RUNX2(+/+) controls, RUNX2(+/m) DPCs presented an impeded progression from the G1 to the S phase in the cell cycle, a lower rate of proliferation, weaker ability of calcification, and distinct ultrastructure. More interestingly, the ultrastructural analysis and energy dispersive X-ray spectrometry (EDS) analysis showed that the CCD tooth exhibited insufficient mineralization of enamel and dentin. This study suggests that the truncated RUNX2 mutant protein may be responsible for the alterations of RUNX2(+/m) DPCs, and RUNX2 gene may be involved in dental development by affecting the cell growth and differentiation, which provides new insights into understanding of dental abnormalities in CCD patients.

[Antisense oligonucleotides targeting protein kinase C alpha inhibits the proliferation of A549 cells].

OBJECTIVE: To investigate the effects of antisense oligonucleotides (ASODN) targeting protein kinase C alpha (PKCalpha) on the proliferation of A549 cells. METHODS: PKCalpha ASODN and random oligonucleotides (RODN) were transfected into A549 cells mediated by polyethyleneimine, and the proliferation and clone formation of A549 cells were detected by CCK-8 and clone formation assay, respectively. The expression of PKCalpha in the transfected cells was analyzed by RT-PCR and Western blotting. RESULTS: Compared with those in the control group, PEI group and PEI-RODN group, the proliferation and clone formation of A549 cells treated with ASODN targeting PKCalpha were significantly inhibited (P<0.05). The expressions of PKCalpha mRNA and protein in PKCalpha ASODN-transfected A549 cells were significantly lower than those in the other 3 groups (P<0.05). CONCLUSION: The PKCalpha ASODN mediated by PEI down-regutates the expression of PKCalpha gene and suppress the proliferation and clone formation of A549 cells.

[Psychological anxiety evaluation and analysis of graduates at a medical university under employment pressure].

OBJECTIVE: To evaluate the psychological anxiety of graduates at a medical university under the ever-increasing employment pressure, so as to provide ground work for psychological intervention on college students. METHODS: Subjects were randomly drawn from the students who majored in clinical medicine, biomedical engineering, nursing and integrated Chinese and western medicine and graduated in the year of 2008 and 2009, with 25 subjects from each major each year, totaling up to 200. In March of their graduation year, they were evaluated by Hamilton Anxiety Scale (HAMA). A general analysis into their anxiety was first made and then the comparative analysis of anxiety on the basis of gender, year group and major of the subjects. RESULTS: Female students showed a significantly higher anxiety than male students. Graduates in 2009 showed a significantly higher anxiety than those in 2008. In terms of the major difference, the anxiety was in a falling curve from integrated Chinese and western medicine, clinical medicine, biomedical engineering and nursing. There was no major difference in the students majoring integrated Chinese and western medicine, clinical medicine and biomedical engineering, but nursing students showed significantly low anxiety. CONCLUSIONS: The increasing employment pressure has caused the significant increase in the anxiety of college students. The employment rate in different majors may play a positive role in anxiety. Generally, female students showed a higher degree of anxiety than male students.

Berberine inhibits cytosolic phospholipase A2 and protects against LPS-induced lung injury and lethality independent of the alpha2-adrenergic receptor in mice.

Acute lung injury is still a significant clinical problem having a high mortality rate despite significant advances in antimicrobial therapy and supportive care made in the past few years. Our previous study demonstrated that berberine (Ber) remarkably decreased mortality and attenuated the lung injury in mice challenged with LPS, but the mechanism behind this remains unclear. Here, we report that pretreatment with Ber significantly reduced pulmonary edema, neutrophil infiltration, and histopathological alterations; inhibited protein expression and phosphorylation of cytosolic phospholipase A2; and decreased thromboxane A2 release induced by LPS. Yohimbine, an alpha2-adrenergic receptor antagonist, did not antagonize these actions of Ber. Furthermore, pretreatment with Ber decreased TNF-alpha production and mortality in mice challenged with LPS, which were enhanced by yohimbine, and Ber combined with yohimbine also improved survival rate in mice subjected to cecal ligation and puncture. Taken together, these observations indicate that Ber attenuates LPS-induced lung injury by inhibiting TNF-alpha production and cytosolic phospholipase A2 expression and activation in an alpha2-adrenoceptor-independent manner. Berberine combined with yohimbine might provide an effective therapeutic approach to acute lung injury during sepsis.