Genome-wide identification of HSF family in peach and functional analysis of PpHSF5 involvement in root and aerial organ development.

BACKGROUND: Heat shock factors (HSFs) play important roles during normal plant growth and development and when plants respond to diverse stressors. Although most studies have focused on the involvement of HSFs in the response to abiotic stresses, especially in model plants, there is little research on their participation in plant growth and development or on the HSF (PpHSF) gene family in peach (Prunus persica). METHODS: DBD (PF00447), the HSF characteristic domain, was used to search the peach genome and identify PpHSFs. Phylogenetic, multiple alignment and motif analyses were conducted using MEGA 6.0, ClustalW and MEME, respectively. The function of PpHSF5 was confirmed by overexpression of PpHSF5 into Arabidopsis. RESULTS: Eighteen PpHSF genes were identified within the peach genome. The PpHSF genes were nonuniformly distributed on the peach chromosomes. Seventeen of the PpHSFs (94.4%) contained one or two introns, except PpHSF18, which contained three introns. The in silico-translated PpHSFs were classified into three classes (PpHSFA, PpHSFB and PpHSFC) based on multiple alignment, motif analysis and phylogenetic comparison with HSFs from Arabidopsis thaliana and Oryza sativa. Dispersed gene duplication (DSD at 67%) mainly contributed to HSF gene family expansion in peach. Promoter analysis showed that the most common cis-elements were the MYB (abiotic stress response), ABRE (ABA-responsive) and MYC (dehydration-responsive) elements. Transcript profiling of 18 PpHSFs showed that the expression trend of PpHSF5 was consistent with shoot length changes in the cultivar 'Zhongyoutao 14'. Further analysis of the PpHSF5 was conducted in 5-year-old peach trees, Nicotiana benthamiana and Arabidopsis thaliana, respectively. Tissue-specific expression analysis showed that PpHSF5 was expressed predominantly in young vegetative organs (leaf and apex). Subcellular localization revealed that PpHSF5 was located in the nucleus in N. benthamiana cells. Two transgenic Arabidopsis lines were obtained that overexpressed PpHSF5. The root length and the number of lateral roots in the transgenic seedlings were significantly less than in WT seedlings and after cultivation for three weeks. The transgenic rosettes were smaller than those of the WT at 2-3 weeks. The two transgenic lines exhibited a dwarf phenotype three weeks after transplanting, although there was no significant difference in the number of internodes. Moreover, the PpHSF5-OE lines exhibited enhanced thermotolerance. These results indicated that PpHSF5 might be act as a suppresser of growth and development of root and aerial organs.

Bufadienolides induce apoptosis and autophagy by inhibiting the AKT signaling pathway in melanoma A­375 cells.

The purpose of the present study was to investigate the effect of bufadienolides on the A­375 melanoma cell line, and to delineate the underlying mechanism. A Cell Counting Kit­8 assay was used to determine the viability of the cells, and flow cytometry was used to evaluate apoptosis. Western blot analysis was used to evaluate the expression levels of proteins involved in the AKT pathway that are associated with apoptosis and autophagy. The results demonstrated that bufadienolides reduced the viability of A­375 cells in a dose­ and a time­dependent manner. Following treatment with bufadienolides, A­375 cells exhibited clear properties that were characteristic of apoptosis and autophagy. The expression levels of the pro­apoptotic proteins Bax and p53 were upregulated, whereas those of the anti­apoptotic proteins, Bcl­2 and caspase­3 were downregulated. In addition, the level of a protein known to be associated with autophagy, microtubule­associated proteins 1A/1B light chain 3­II, was increased, whereas that of p62 protein was reduced. Finally, the AKT signaling pathway was blocked in the bufadienolide­treated A­375 cells. In conclusion, these results revealed that bufadienolides effectively induced apoptosis and autophagy in A­375 cells via the AKT pathway, and therefore may be one of the candidate targets for the future development of targeted drugs to treat melanoma.

Baicalin potentiates TRAIL­induced apoptosis through p38 MAPK activation and intracellular reactive oxygen species production.

The combination of tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) with other agents has been recognized as a promising strategy to overcome TRAIL resistance in cancer cells. Baicalin (5, 6­dihydroxy­7­o­glucuronide flavone) is a flavonoid from the root of the medicinal herb Scutellaria baicalensis Georgi, which has been reported to exert antioxidant, anti­inflammatory, antiviral and anticancer activities in vitro. However, the effect of baicalin on TRAIL­induced cytotoxicity has not been previously reported. In the present study, the effect of combining TRAIL and baicalin was investigated in non­small cell lung cancer cell lines. The results revealed that baicalin was able to sensitize A549 and H2009 cells to TRAIL­induced apoptosis. This was detected by the potentiation of poly­adenosine­5'­diphosphate­ribose polymerase cleavage and Annexin V­fluorescein isothiocyanate staining of cells co­treated with baicalin and TRAIL. In addition, p38 mitogen­activated protein kinase was activated in baicalin and TRAIL co­treated cancer cells, whereas the p38 inhibitor SB203580 effectively suppressed cell death within the co­treated cells. Butylated hydroxyanisole and N­acetyl­cysteine, known reactive oxygen species (ROS) scavengers, significantly suppressed the potentiated cytotoxicity induced by baicalin and TRAIL co­treatment. The present study is the first, to the best of our knowledge, to demonstrate that baicalin enhances the anticancer activity of TRAIL via p38 activation and ROS accumulation, and may be exploited for anticancer therapy.