Identification of HuSPL family and key role of HuSPL12 in regulation of betalain biosynthesis in pitaya.

The SQUAMOSA promoter binding protein-like (SPL) gene family is a unique family of plant-specific transcription factors (TFs), which plays vital roles in a variety of plant biological processes. Its role in betalain biosynthesis in Hylocereus undantus; however, is still unclear. Here, we report a total of 16 HuSPL genes from the pitaya genome, which were unevenly distributed among nine chromosomes. The HuSPL genes were clustered into seven groups, and most HuSPLs within the same group shared similar exon-intron structures and conserved motifs. Eight segment replication events in the HuSPL gene family were the main driving force behind the gene family expansion. Nine of the HuSPL genes had potential target sites for Hmo-miR156/157b. Hmo-miR156/157b-targeted HuSPLs exhibited differential expression patterns compared with constitutive expression patterns of most Hmo-miR156/157b-nontargeted HuSPLs. The expression of Hmo-miR156/157b gradually increased during fruit maturation, while the expression of Hmo-miR156/157b-targeted HuSPL5/11/14 gradually decreased. In addition, the lowest expression level of Hmo-miR156/157b-targeted HuSPL12 was detected 23rd day after flowering, when the middle pulps started to turn red. HuSPL5, HuSPL11, HuSPL12, and HuSPL14 were nucleus-localized proteins. HuSPL12 could inhibit the expression of HuWRKY40 by binding to its promoter. Results from yeast two-hybrid and bimolecular fluorescence complementation assays showed that HuSPL12 could interact with HuMYB1, HuMYB132, or HuWRKY42 TFs responsible for betalain biosynthesis. The results of the present study provide an essential basis for future regulation of betalain accumulation in pitaya.

Identification of HuSWEET Family in Pitaya (Hylocereus undatus) and Key Roles of HuSWEET12a and HuSWEET13d in Sugar Accumulation.

The sugar composition and content of fruit have a significant impact on their flavor and taste. In pitaya, or dragon fruit, sweetness is a crucial determinant of fruit taste and consumer preference. The sugars will eventually be exported transporters (SWEETs), a novel group of sugar transporters that have various physiological functions, including phloem loading, seed filling, nectar secretion, and fruit development. However, the role of SWEETs in sugar accumulation in pitaya fruit is not yet clear. Here, we identified 19 potential members (HuSWEET genes) of the SWEET family in pitaya and analyzed their conserved motifs, physiochemical characteristics, chromosomal distribution, gene structure, and phylogenetic relationship. Seven highly conserved α-helical transmembrane domains (7-TMs) were found, and the HuSWEET proteins can be divided into three clades based on the phylogenetic analysis. Interestingly, we found two HuSWEET genes, HuSWEET12a and HuSWEET13d, that showed strong preferential expressions in fruits and an upward trend during fruit maturation, suggesting they have key roles in sugar accumulation in pitaya. This can be further roughly demonstrated by the fact that transgenic tomato plants overexpressing HuSWEET12a/13d accumulated high levels of sugar in the mature fruit. Together, our result provides new insights into the regulation of sugar accumulation by SWEET family genes in pitaya fruit, which also set a crucial basis for the further functional study of the HuSWEETs.

Dimethyl celecoxib sensitizes gastric cancer cells to ABT-737 via AIF nuclear translocation.

Gastric cancer is the fourth most common cancer in the world. The clinical applications of both chemotherapy and targeted drugs are limited because of the complexity of gastric cancer. In this study, sulforhodamine B, colony formation assay, 4',6-diamidino-2-phenylindole (DAPI) stain, flow cytometry were used to determine the in vitro cytotoxicity, apoptosis and mitochondrial membrane potential of gastric cancer AGS and HGC-27 cells before and after treatment. Real-time PCR and Western blot were used to analyse the mRNA transcription and protein expression respectively. Confocal microscopy was used to determine the localization of target protein within the cells. Treatment with the combination of ABT-737 and 2,5-dimethyl-celecoxib (DMC) showed strong synergistic effect in both AGS and HGC-27 cells. Moreover, DMC would not influence the intracellular prostaglandin E2 (PGE2) level, thus lacking the toxicity profile of celecoxib. Interestingly, given the significant synergistic effect, combination treatment did not affect the protein expression of BH-3 proteins including Puma, Noxa and Bim. In combination treatment, cell apoptosis was found independent of caspase-3 activation. The translocation of apoptosis-inducing factor (AIF) from mitochondrion to nuclear was responsible for the induced apoptosis in the combination treatment. Taken together, this study provided a novel combination treatment regimen for gastric cancer. Furthermore, the existence of caspase-independent apoptotic pathway induced by treatment of ABT-737 was not yet seen until combined with DMC, which shed light on an alternative mechanism involved in Bcl-2 inhibitor-induced apoptosis.

Deacetylisovaltratum disrupts microtubule dynamics and causes G2/M-phase arrest in human gastric cancer cells in vitro.

AIM: Deacetylisovaltratum (DI) is isolated from the traditional Chinese herbal medicine Patrinia heterophylla Bunge, which exhibits anti-cancer activity. Here, we investigated the effects of DI on human gastric carcinoma cell lines in vitro and elucidated its anti-cancer mechanisms. METHODS: Human gastric carcinoma AGS and HGC-27 cell lines were treated with DI, and cell viability was detected with MTT assay. Cell cycle stages, apoptosis and mitochondrial membrane potential were measured using flow cytometry. Protein levels were analyzed by Western blotting. Tubulin polymerization assays and immunofluorescence were used to characterize the tubulin polymerization process. RESULTS: DI inhibited the cell viability of AGS and HGC-27 cells in a dose- and time-dependent manner with IC50 values of 12.0 and 28.8 µmol/L, respectively, at 24 h of treatment. Treatment with DI (10-100 µmol/L) dose-dependently promoted tubulin polymerization, and induced significant G2/M cell cycle arrest in AGS and HGC-27 cells. Moreover, DI treatment disrupted mitochondrial membrane potential and induced caspase-dependent apoptosis in AGS and HGC-27 cells. CONCLUSION: DI induces G2/M-phase arrest by disrupting tubulin polymerization in human gastric cancer cells, which highlights its potent anti-cancer activity and potential application in gastric cancer therapy.

Identification of HubHLH family and key role of HubHLH159 in betalain biosynthesis by activating the transcription of HuADH1, HuCYP76AD1-1, and HuDODA1 in pitaya.

Basic helix-loop-helix (bHLH) proteins are dimeric transcription factors (TFs) involved in various plant physiological and biological processes. Despite this, little is known about the molecular properties and roles of bHLH TFs in pitaya betalain biosynthesis. Here we report the identification of 165 HubHLH genes in H. undantus genome, their chromosomal distribution, physiochemical characteristics, conserved motifs, gene structure, phylogeny and synteny of HubHLH genes. Based on phylogenetic relationship analysis, the 165 HubHLHs were divided into 26 subfamilies and unequally distributed on the 11 chromosomes of pitaya. Based on the pitaya transcriptome data, a candidate gene HubHLH159 was obtained, and the real-time quantitative PCR analysis confirmed that HubHLH159 showed a high expression level in 'Guanhuahong' pitaya (red-pulp) at mature stage, indicating its role in betalain biosynthesis. HubHLH159 is a Group II protein and contains a bHLH domain. It is a nuclear protein with transcriptional activation activity. Dual luciferase reporter assays and virus-induced gene silencing (VIGS) experiments showed that HubHLH159 promotes betalain biosynthesis by activating the expression of HuADH1, HuCYP76AD1-1, and HuDODA1. The results of the present study lay a new theoretical reference for the regulation of pitaya betalain biosynthesis and also provides as essential basis for the future analysis of the functions of HubHLH gene family.

Ethyl acetate extract from Inula helenium L. inhibits the proliferation of pancreatic cancer cells by regulating the STAT3/AKT pathway.

Sesquiterpene lactones are bioactive compounds that have been identified as responsible for the anticancer activity of the medicinal herb, Inula helenium L. (IHL). However, the mechanisms of action involved in the anti­pancreatic cancer activity of IHL have yet to be elucidated. The present study used an optimized extraction strategy to obtain sesquiterpene lactones from IHL (the resulting product termed ethyl acetate extract of IHL; EEIHL), and examined the potential mechanisms involved in the anti­pancreatic cancer activity of EEIHL. Ethanol and ethyl acetate were used to extract sesquiterpene lactones from IHL to give the final product EEIHL. Cell Counting Kit­8, colony formation and Annexin V/propidium iodide assays were used to detect the anti­proliferative activity of EEIHL. Cell migration was determined with a wound healing assay. mRNA and protein expression levels were analyzed by reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. It was identified that low concentrations of EEIHL caused CFPAC­1 cell cycle arrest in the G0/G1 phase, whereas high concentrations of EEIHL induced mitochondria­dependent apoptosis. In addition, EEIHL could inhibit the phosphorylation of the signal transducer and activator of transcription (STAT)3/AKT pathway, potentially resulting in impeded cell mobility. In conclusion, EEIHL could activate mitochondrial­dependent apoptosis and inhibit cell migration through the STAT3/AKT pathway in CFPAC-1 cells.