A PpEIL2/3-PpNAC1-PpWRKY14 module regulates fruit ripening by modulating ethylene production in peach.

WRKY transcription factors play key roles in plant resistance to various stresses, but their roles in fruit ripening remain largely unknown. Here, we report a WRKY gene PpWRKY14 involved in the regulation of fruit ripening in peach. The expression of PpWRKY14 showed an increasing trend throughout fruit development. PpWRKY14 was a target gene of PpNAC1, a master regulator of peach fruit ripening. PpWRKY14 could directly bind to the promoters of PpACS1 and PpACO1 to induce their expression, and this induction was greatly enhanced when PpWRKY14 formed a dimer with PpNAC1. However, the transcription of PpNAC1 could be directly suppressed by two EIN3/EIL1 genes, PpEIL2 and PpEIL3. The PpEIL2/3 genes were highly expressed at the early stages of fruit development, but their expression was programmed to decrease significantly during the ripening stage, thus derepressing the expression of PpNAC1. These results suggested a PpEIL2/3-PpNAC1-PpWRKY14 module that regulates fruit ripening by modulating ethylene production in peach. Our results provided an insight into the regulatory roles of EIN3/EIL1 and WRKY genes in fruit ripening.

Association between personality traits and psychological distress among postmenopausal women with coronary heart disease: A cross-sectional survey and mediation analysis.

Postmenopausal women with negative personality characteristics are at an increased risk of psychological disorders, yet little is known about the mechanism underlying the relationship between type D personality and psychological distress in postmenopausal women with coronary disease. This study assessed the mediating roles of perceived social support and self-perceived burden in the relationship between type D and psychological distress based on the equity theory and stress-buffering model. Demographic characteristics, type D, psychological distress, perceived social support, and self-perceived burden were completed by 335 participants with self-reported questionnaires using a cross-sectional design in Southeast China. The results revealed that perceived social support and self-perceived burden both separately and serially mediated the relationship between type D personality and psychological distress. Effective intervention strategies aimed at improving perceived social support or reducing self-perceived burden may be beneficial in reducing psychological distress.

The relationship between filial values and preparedness in adult -child caregivers of patients with chronic heart failure: A cross-sectional study.

OBJECTIVES: To explore the relationship between filial values (responsibility, reciprocity, and obligation) and caregiving preparedness in adult child carers of patients with chronic heart failure (CHF). METHODS: A total of 339 adult child caregivers of CHF patients were recruited from six hospitals in China. Self-report questionnaires were used to measure caregivers' filial values and their caregiving preparedness. Descriptive analysis, Spearman's correlation analysis, and hierarchical regression analysis were performed. The study adhered to the STROBE guidelines. RESULTS: Filial values were positively correlated with caregiving preparedness. Hierarchical regression analysis revealed a positive relationship between the combined variables of care and respect with caregiver preparedness, jointly explaining 7.9 % of the variance. CONCLUSIONS: Filial values promote caregiver preparedness, broadening the applicability of the Caregiver Empowerment Model.

Return to Work Experience of Young and Middle-Aged Patients With Acute Myocardial Infarction: A Longitudinal Qualitative Study.

BACKGROUND: Return to work (RTW) is a critical component of rehabilitation for most young and middle-aged patients after an acute myocardial infarction (AMI). Its success is related to the quality of life and social psychological function of patients, and their social economic growth. However, healthcare professionals often do not deeply understand the patients' experience and their difficulties and coping methods during this process, which limits their ability to institute effective management and support. OBJECTIVE: In this study, we aimed to explore the lived experiences and change processes of young and middle-aged patients with AMI at the different stages of RTW. METHODS: A descriptive qualitative approach was used. Patients aged 20 to 59 years with AMI were recruited from the Department of Cardiology of 3 general hospitals. Data were collected via semistructured interviews. Data analysis was performed by conventional content analysis methods. RESULTS: In total, 18 participants were included. Five main themes emerged: (1) "chaos," (2) "rebuilding," (3) "conflict," (4) "coping," and (5) "benefits." Patients may be more concerned about physical recovery during the initial clinical event. They then begin to plan and adjust for an RTW. Patients in the maintenance phase need strategies to prevent, identify, and respond to conflicts and challenges to maintain long-term stable work. CONCLUSION: We identified several post-AMI stages spanning from the initial illness event to the maintenance of stable work. We described their perceived barriers, coping strategies, and support needs at these various stages. These data are crucial for healthcare professionals to develop improved vocational rehabilitation strategies for patients with AMI.

Two R2R3-MYB genes cooperatively control trichome development and cuticular wax biosynthesis in Prunus persica.

The fruit surface has an enormous impact on the external appearance and postharvest shelf-life of fruit. Here, we report two functionally redundant genes, PpMYB25 and PpMYB26, involved in regulation of fruit skin texture in peach. PpMYB25 can activate transcription of PpMYB26 and they both induce trichome development and cuticular wax accumulation, resulting in peach fruit with a fuzzy and dull appearance. By contrast, nonfunctional mutation of PpMYB25 caused by an insertional retrotransposon in the last exon in nectarine fails to activate transcription of PpMYB26, resulting in nectarine fruit with a smooth and shiny appearance due to loss of trichome initiation and decreased cuticular wax accumulation. Secondary cell wall biosynthesis in peach fruit pubescence is controlled by a transcriptional regulatory network, including the master regulator PpNAC43 and its downstream MYB transcription factors such as PpMYB42, PpMYB46 and PpMYB83. Our results show that PpMYB25 and PpMYB26 coordinately regulate fruit pubescence and cuticular wax accumulation and their simultaneous perturbation results in the origin of nectarine, which is botanically classified as a subspecies of peach.

Floral homeotic proteins modulate the genetic program for leaf development to suppress trichome formation in flowers.

As originally proposed by Goethe in 1790, floral organs are derived from leaf-like structures. The conversion of leaves into different types of floral organ is mediated by floral homeotic proteins, which, as described by the ABCE model of flower development, act in a combinatorial manner. However, how these transcription factors bring about this transformation process is not well understood. We have previously shown that floral homeotic proteins are involved in suppressing the formation of branched trichomes, a hallmark of leaf development, on reproductive floral organs of Arabidopsis Here, we present evidence that the activities of the C function gene AGAMOUS (AG) and the related SHATTERPROOF1/2 genes are superimposed onto the regulatory network that controls the distribution of trichome formation in an age-dependent manner. We show that AG regulates cytokinin responses and genetically interacts with the organ polarity gene KANADI1 to suppress trichome initiation on gynoecia. Thus, our results show that parts of the genetic program for leaf development remain active during flower formation but have been partially rewired through the activities of the floral homeotic proteins.

The MADS-box gene PpPI is a key regulator of the double-flower trait in peach.

Double flower is an invaluable trait in ornamental peach, but the mechanism underlying its development remains largely unknown. Here, we report the roles of ABCE model genes in double flower development in peach. A total of nine ABCE regulatory genes, including eight MADS-box genes and one AP2/EREBP gene, were identified in the peach genome. Subcellular localization assay showed that all the ABCE proteins were localized in the nucleus. Four genes, PpAP1, PpAP3, PpSEP3, and PpPI, showed a difference in expression levels between single and double flowers. Ectopic overexpression of PpPI increased petal number in Arabidopsis, while transgenic lines overexpressing PpAP3 or PpSEP3 were morphologically similar to wild-type. Ectopic overexpression of PpAP1 resulted in a significant decrease in the number of basal leaves and caused early flowering. These results suggest that PpPI is likely crucial for double flower development in peach. In addition, double flowers have petaloid sepals and stamens, and single flower could occasionally change to be double flower by converting stamens to petals in peach, suggesting that the double-flower trait is likely to have evolved from an ancestral single-flower structure. Our results provide new insights into mechanisms underlying the double-flower trait in peach.

The sucrose transporter MdSUT4.1 participates in the regulation of fruit sugar accumulation in apple.

BACKGROUND: Sugar content is an important determinant of fruit sweetness, but details on the complex molecular mechanism underlying fruit sugar accumulation remain scarce. Here, we report the role of sucrose transporter (SUT) family in regulating fruit sugar accumulation in apple. RESULTS: Gene-tagged markers were developed to conduct candidate gene-based association study, and an SUT4 member MdSUT4.1 was found to be significantly associated with fruit sugar accumulation. MdSUT4.1 encodes a tonoplast localized protein and its expression level had a negative correlation with fruit sugar content. Overexpression of MdSUT4.1 in strawberry and apple callus had an overall negative impact on sugar accumulation, suggesting that it functions to remobilize sugar out of the vacuole. In addition, MdSUT4.1 is located on chromosomal region harboring a previously reported QTL for sugar content, suggesting that it is a candidate gene for fruit sugar accumulation in apple. CONCLUSIONS: MdSUT4.1 is involved in the regulation of fruit sugar accumulation in apple. This study is not only helpful for understanding the complex mechanism of fruit sugar accumulation, but it also provides molecular tools for genetic improvement of fruit quality in breeding programs of apple.

The modification of mitochondrial energy metabolism and histone of goat somatic cells under small molecules compounds induction.

In recent years, induced pluripotent stem cells (iPSCs) technique is able to allow us to generate pluripotency from somatic cells in vitro through the over expression of several transcription factors. Normally, viral vectors and transcription factors are commonly used on iPSC technique, which could cause many barriers on further application. In this study, we attempt to process a new method to obtain pluripotency from goat somatic cells in vitro under fully chemically defined condition. The results showed that chemically induced pluripotent stem cells-like cells (CiPSC-like cells) colonies were generated from goat ear fibroblasts by fully small-molecule compounds. Those three dimensions colonies were similar with mouse iPSCs in morphology and had strong positive alkaline phosphatase (AP) activity and expressed pluripotency related genes OCT4, SOX2, NANOG, CDH1, TDGF, GDF3, DAX1, REX1, which determined by RT-PCR. Those colonies could also differentiate into different cell types derived from three germ layers proved by RT-PCR and immunofluorescence assays. The expression of glycolysis-related genes about PGAM1, KPYM2 and HXK2 in CiPSC-like colonies formation groups was significantly higher than their parental fibroblasts, but not in the non-CiPSC-like colonies formation group. The expression of histone acetylation and methylation-related genes, HAT1 and SMYD3, was not significantly up-regulated within different groups compared to their parental fibroblasts, respectively. Yet, the expression of histone methylation-related gene, KDM5B, was significantly up-regulated on the cells from non-colonies formation group compared to parental fibroblasts, but the expression of KDM5B of the cells from CiPSC-like cell colonies was not significantly difference compared to that of parental fibroblasts. In conclusion, this is the first report that CiPSC-like cells could be generated in vitro from goat rather than just mouse under fully chemically defined condition. The generation of CiPSC-like colonies may be depended on the correct modification of energy metabolism and histone epigenetic during the reprogramming, rather than just the over-expression of those pluripotency-related genes. This study will strongly support us to further establish the stable goat CiPSC lines without any integration of exogenous genes.

Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation.

The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL is currently unknown. To better understand the regulatory relationship between LFY and AP1/CAL and to obtain deeper insights into the control of floral initiation, we monitored the activity of LFY in the absence of AP1/CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL act as part of an incoherent feed-forward loop, a network motif where two interconnected pathways or transcription factors act in opposite directions on a target gene, to control the establishment of a stable developmental program for the formation of flowers.

The radiotherapy-sensitization effect of cantharidin: Mechanisms involving cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair.

BACKGROUND: Cantharidin is an inhibitor of protein phosphatase 2 A (PP2A), and has been frequently used in clinical practice. In our previous study, we proved that cantharidin could arrest cell cycle in G2/M phase. Since cells at G2/M phase are sensitive to radiotherapy, in the present study, we investigated the radiotherapy-sesitization effect of cantharidin and the potential mechanisms involved. METHODS: Cell growth was determined by MTT assay. Cell cycle was evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. Expression of mRNA was tested by microarray assay and real-time PCR. Clinical information and RNA-Seq expression data were derived from The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. Survival analysis was obtained by Kaplan-Meier estimates. RESULTS: Cantharidin strengthened the growth inhibition effect of irradiation. Cantharidin drove pancreatic cancer cells out of quiescent G0/G1 phase and arrested cell cycle in G2/M phase. As a result, cantharidin strengthened DNA damage which was induced by irradiation. Moreover, cantharidin repressed expressions of several genes participating in DNA damage repair, including UBE2T, RPA1, GTF2HH5, LIG1, POLD3, RMI2, XRCC1, PRKDC, FANC1, FAAP100, RAD50, RAD51D, RAD51B and DMC1, through JNK, ERK, PKC, p38 and/or NF-κB pathway dependent manners. Among these genes, worse overall survival for pancreatic cancer patients were associated with high mRNA expressions of POLD3, RMI2, PRKDC, FANC1, RAD50 and RAD51B, all of which could be down-regulated by cantharidin. CONCLUSION: Cantharidin can sensitize pancreatic cancer cells to radiotherapy. Multiple mechanisms, including cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair, may be involved.

Early development of porcine parthenogenetic embryos and reduced expression of primed pluripotent marker genes under the effect of lysophosphatidic acid.

To further promote the early development of porcine embryos and capture "naïve" pluripotent state within blastocyst, the experiment explored the effects of lysophosphatidic acid (LPA) on the early development of porcine parthenogenetic embryos and the expression of pluripotency relevant genes. The results showed that the addition of 50 µM LPA significantly improved parthenogenetic embryo cleavage rate (82.7% vs. 74.7%, p < 0.05), blastocyst rate (24.5% vs. 11.3%, p < 0.05) and blastocyst cell count (56 ± 7.9 vs. 42 ± 1.0, p < 0.05) than that of the control group. In addition, immunostaining experiment determined that the fluorescence intensity of OCT4 was also significantly higher than that of the control group. The quantitative real-time polymerase chain reaction (qRT-PCR) test revealed that addition of 50 µM LPA could significantly enhance the expression level of pluripotent gene OCT4 and trophoblast marker genes CDX2, however, decrease the expression of primitive hypoblast marker gene GATA4. The results also indicated that LPA might decrease the expression of GATA4 through the ROCK signalling pathway. For further investigating the effect of the addition of LPA on the expression of "primed" and "naïve" genes, we also detected the expression of those pluripotency-related genes by qRT-PCR. The results showed addition of LPA had no significant effect on the expression of "naïve" pluripotent genes, but it was able to significantly decrease the expression of "primed" pluripotent genes, NODAL and Activin-A; furthermore, it also could significantly improve the expression of OCT4 and c-Myc which act as two important ES cell renewal factors. Above all, the addition of LPA can facilitate the early development of porcine parthenogenetic embryos, which may be able to benefit for capturing "naïve" pluripotency in vitro through inhibiting "primed" pluripotency.

High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis).

BACKGROUND: G1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from "Guoqing No.1" (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development. RESULTS: A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5' RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression. CONCLUSION: Our study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.

3ß-Angeloyloxy-8ß,10ß-dihydroxyeremophila-7(11)-en-12,8α-lactone Inhibits Lipopolysaccharide-Induced Nitric Oxide Production in RAW264.7 Cells.

Farfugium japonicum (L.) KITAM, named "Lian-Peng-Cao" in China, has been traditionally used in Chinese folk medicine to treat sore throat, cold and cough due to its anti-inflammatory properties. In this study, the anti-inflammatory action of 3ß-angeloyloxy-8ß,10ß-dihydroxyeremophila-7(11)-en-12,8α-lactone (FJ1) isolated from Farfugium japonicum and its molecular mechanism in RAW264.7 cells were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that FJ1 with or without 3 µg/mL lipopolysaccharide (LPS) had no significant cytotoxicity in RAW264.7 cells. The production of nitric oxide (NO) was identified with a Griess reagent kit. The mRNA expression of inducible nitric oxide synthase (iNOS) and cytokines, including tumor necrosis factor α (TNF-α) and cyclooxygenase-2 (COX-2), was measured by real-time polymerase chain reaction (PCR). Reactive oxygen species (ROS) production was detected by flow cytometry analysis. Western blot was used to examine the protein expression of nuclear factor-kappa B (NF-κB)/p65, inhibitor of kappa B (IκB)-α, phosphorylated IκB-α (p-IκB-α), mitogen-activated protein kinase (MAPK) molecules, iNOS, and TNF-α. We discovered that FJ1 possesses anti-inflammatory effects that inhibit the release of LPS-stimulated pro-inflammatory cytokines, including NO and ROS. The molecular mechanism of FJ1-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules, including extracellular signal-related kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), FJ1 also reverses IκB degradation and attenuates the mRNA and protein expression of NF-κB-related downstream inducible enzymes and cytokines, such as iNOS, TNF-α in RAW264.7 cells. The results suggest that FJ1 has anti-inflammatory properties, which indicates that F. japonicum can be utilized to treat inflammatory diseases. The potential mechanism is associated with the NF-κB and MAPK activation pathways in LPS-stimulated macrophages.

iTRAQ-based quantitative proteomics analysis revealed alterations of carbohydrate metabolism pathways and mitochondrial proteins in a male sterile cybrid pummelo.

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway.

AIM: Telekin, isolated from the Chinese herb Carpesium divaricatum, has shown anti-proliferation effects against various cancer cells, including hepatocellular carcinoma cells. In this study, we investigated the anti-proliferation mechanisms of telekin in human hepatocellular carcinoma HepG2 cells in vitro. METHODS: HepG2 cells were treated with telekin. Cell viability was evaluated using MTT assay. Flow cytometry was used to measure cell cycle profiles, ROS level and apoptosis. The protein expression levels were analyzed with Western blotting. RESULTS: Telekin (3.75-30 µmol/L) dose-dependently inhibited the viability of HepG2 cells and induced l apoptosis. Furthermore, the treatment induced cell cycle arrest at G2/M phase, accompanied by significantly increased the phosphorylation of Cdc25A and Cdc2, and decreased Cyclin B1 level. Moreover, the treatment significantly stimulated ROS production, and increased the phosphorylation of p38 and MAPKAPK-2 in the cells. Pretreatment with the antioxidant NAC (2.5, 5, and 10 mmol/L), or the p38 MAPK inhibitor SB203580 (2.5 and 5 µmol/L) dose-dependently attenuated these telekin-induced effects in the cells. CONCLUSION: Telekin suppresses hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via activating the p38 MAPK pathway.

Metabolic basis for superior antioxidant capacity of red-fleshed peaches.

Peach fruit is an important natural source of phenolic compounds that are well-known to have health benefits, but their metabolic basis remain elusive. Here, we report on phenolic compounds accumulation and antioxidant activity of ripe fruits in peach. A considerable variation in phenolic compounds content was observed among peach germplasm, with significantly higher levels detected in red-fleshed peaches compared to non-red-fleshed peaches. Antioxidant activity of crude extracts from ripe fruits showed significant differences among peach germplasm, with red-fleshed peaches having the strongest antioxidant activity. Intriguingly, it was observed that total phenolics instead of anthocyanins were strongly associated with antioxidant activity. Phenolic compounds content and antioxidant activity showed dynamic changes throughout fruit development, and these were much higher in the peel than in the flesh. Metabolomic analysis unveiled a coordinated accumulation of anthocyanins as well as key components of flavonoids and phenolic acids, which endows red-fleshed peaches with superior antioxidant activity.

Proteomic analysis on salicylic acid-induced salt tolerance in common wheat seedlings (Triticum aestivum L.).

The influence of salicylic acid (SA) on the salt tolerance mechanism in seedlings of common wheat (Triticum aestivum L.) was investigated using physiological measurements combined with global expression profiling (proteomics). In the present study, 0.5mM SA significantly reduced NaCl-induced growth inhibition in wheat seedlings, manifesting as increased fresh weights, dry weights, and photosynthetic pigments, but decreased lipid peroxidation. Two-week-old wheat seedlings treated with 0.5mM SA, 250 mM NaCl and 250 mM NaCl+0.5mM SA for 3 days were used for the proteomic analyses. In total, 39 proteins differentially regulated by both salt and SA were revealed by 2D PAGE, and 38 proteins were identified by MALDI-TOF/TOF MS. The identified proteins were involved in various cellular responses and metabolic processes including signal transduction, stress defense, energy, metabolism, photosynthesis, and others of unknown function. All protein spots involved in signal transduction and the defense response were significantly upregulated by SA under salt stress, suggesting that these proteins could play a role in the SA-induced salt resistance in wheat seedlings.

1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro.

AIM: To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL), a novel eudesmane-type sesquiterpene isolated from Aster himalaicus, on the cell cycle and apoptosis in human glioblastoma cells in vitro. METHODS: Human malignant glioblastoma cell lines U87 and A172 were used. The cytotoxicity of OEL was examined using the MTT assay. Cell apoptosis was assessed with DAPI staining and flow cytometry. DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting. Cell cycle profiles were measured with flow cytometry. The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR. The protein expression of γ-H2AX, caspase-9, caspase-3, p53, p21Waf1/Cip1, cyclin B1, and cdc2 was analyzed with Western blotting. RESULTS: Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose- and time-dependent manners (the values of IC(50) at 48 and 72 h were 29.5 and 16.99 µmol/L, respectively, in U87 cells; 7.2 and 9.5 µmol/L, respectively, in A172 cells). OEL (10-30 µmol/L) induced apoptosis and G(2)/M phase arrest in both U87 and A172 cells. OEL induced the phosphorylation of cdc2, a G(2)/M phase cyclin-dependent kinase, and decreased the expression of cyclin B1 required for progression through the G(2)/M phase in U87 cells. The compound remarkably increased the phosphorylation of H2AX in U87 cells. Moreover, OEL increased the mRNA and protein levels of p53 and its target gene p21(Waf1/Cip1) in U87 cells. The compound also induced p53 phosphorylation. Pretreatment with PFT-α, a specific inhibitor of p53 transcriptional activity, could partially reverse the inhibition of OEL on the viability of U87 and A172 cells. CONCLUSION: OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage, p53-mediated cell cycle arrest and apoptosis, thus warrants further studies as a lead compound of anti-glioblastoma drug.