Involvement of LeMDR, an ATP-binding cassette protein gene, in shikonin transport and biosynthesis in Lithospermum erythrorhizon.

BACKGROUND: Shikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism. RESULTS: We successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis. CONCLUSIONS: Our findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin.

A Classic Near-Infrared Probe Indocyanine Green for Detecting Singlet Oxygen.

The revelation of mechanisms of photodynamic therapy (PDT) at the cellular level as well as singlet oxygen (¹O2) as a second messengers requires the quantification of intracellular ¹O2. To detect singlet oxygen, directly measuring the phosphorescence emitted from ¹O2 at 1270 nm is simple but limited for the low quantum yield and intrinsic efficiency of ¹O2 emission. Another method is chemically trapping ¹O2 and measuring fluorescence, absorption and Electron Spin Resonance (ESR). In this paper, we used indocyanine green (ICG), the only near-infrared (NIR) probe approved by the Food and Drug Administration (FDA), to detect ¹O2 in vitro. Once it reacts with ¹O2, ICG is decomposed and its UV absorption at 780 nm decreases with the laser irradiation. Our data demonstrated that ICG could be more sensitive and accurate than Singlet Oxygen Sensor Green reagent(®) (SOSG, a commercialized fluorescence probe) in vitro, moreover, ICG functioned with Eosin Y while SOSG failed. Thus, ICG would reasonably provide the possibility to sense ¹O2 in vitro, with high sensitivity, selectivity and suitability to most photosensitizers.

A potent anticancer agent of shikonin derivative targeting tubulin.

In this study, a shikonin ester derivative, compound , was selected to evaluate its anticancer activities and we found that compound exhibited better antitubulin activities against the human HepG2 cell line with an IC50 value of 1.097 µM. Furthermore, the inhibition of tubulin polymerization results indicated that compound demonstrated the most potent antitubulin activity (IC50 = 13.88), which was compared with shikonin and colchicine as positive controls (IC50 = 25.28 µM and 22.56 µM), respectively. Compound was simulated to have good binding site with tubulin and arrested the cell cycle at G2/M phase, which also induces apoptosis in HepG2 cells, in which P53 and members of Bcl-2 protein family were both involved in the progress of apoptosis revealed by western blot. Confocal microscopy observations revealed compound targeted tubulin and altered its polymerization by interfering with microtubule organization. Based on these results, compound functions as a potent anticancer agent targeting tubulin.

[Symbiotic efficiency and genetic diversity of the rhizobia isolated from Leucaena leucocephala in Liangshan Prefecture].

OBJECTIVE: We analyzed the symbiotic efficiency and genetic diversity of rhizobia isolated from Leucaena leucocephala in Liangshan Prefecture of SichuanProvince. METHODS: We studied genetic diversity of these isolates with 16S rRNA RFLP, BOX-PCR and AFLP fingerprinting, and constructed phylogenetic tree based on the concatenated sequences of the four housekeeping genes 16S rRNA, recA, atpD and glnII. The nodulation ability and the symbiotic efficiency of the isolates were tested by plant inoculation assay on their original host plant. RESULTS: Genetic diversity and phylogenetic tree indicate that 26 isolates were assigned as Sinorhizobium, 3 Bradyrhizobium, 3 Rhizobium and 1 Mesorhizobium. SCAU203 might represent a new Rhizobium group, SCAU211 might represent a new Bradyrhizobium group, the other three representative strains were located in three phylogenic branches and closely related to S. americanum, M. plurifarium and R. huautlense, respectively. In the nodulation and symbiotic efficiency assay, only 2 of the 20 isolates promoted the growth of L. leucocephala, but 3 isolates had a growth slowing effect on the host, while the other isolates (84%) were ineffective on symbiotic nitrogen fixation. CONCLUSION: The majority of rhizobia isolated from L. leucocephala in Liangshan Prefecture were ineffective on symbiotic nitrogen fixation.

Exploring the evolutionary process of alkannin/shikonin O-acyltransferases by a reliable Lithospermum erythrorhizon genome.

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contigN50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon's roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT's functional differentiation in evolution.

Comparative genome/transcriptome analysis probes Boraginales' phylogenetic position, WGDs in Boraginales, and key enzyme genes in the alkannin/shikonin core pathway.

Boraginales (the forget-me-not order) is a core group within the lamiids clade. However, until now, no genome from Boraginales has been reported, and published transcriptomes are also rare. Here, we report the first Boraginales species de novo genome (i.e. Echium plantagineum genome) and seven other Boraginales species transcriptomes to probe three issues: (i) Boraginales' phylogenetic position within the lamiids clade; (ii) potential whole genome duplications (WGDs) in Boraginales; and (iii) candidate key enzyme genes in the alkannin/shikonin core pathway. The results showed that: (i) Boraginales was most probably closer to the Solanales/Gentianales clade than the Lamiales clade, at least based on the single-copy orthologous genes from genome/transcriptome data; (ii) after the gamma (γ) event, Boraginaceae (classified into the Boraginales I clade) probably underwent at least two rounds of WGD, whereas Heliotropiaceae and Ehretiaceae (classified into the Boraginales II clade) probably underwent only one round of WGD; and (iii) several candidate key enzyme genes in the alkannin/shikonin core pathway were inferred, e.g. genes corresponding to geranyl cyclase, naphthol hydroxylase and O-acyl transferase.

Naphthoquinones: A continuing source for discovery of therapeutic antineoplastic agents.

Naturally occurring naphthoquinones, usually in forms of botanical extracts, have been implicated with human life since ancient time, far earlier than their isolation and identification in modern era. The long use history of naphthoquinones has witnessed their functional shift from the original purposes as dyes and ornaments toward medicinal benefits. Hitherto, numerous studies have been carried out to elucidate the pharmacological profile of both natural and artificial naphthoquinones. A number of entities have been identified with promising therapeutic potential. Apart from the traditional effects of wound healing, anti-inflammatory, hemostatic, antifertility, insecticidal and antimicrobial, etc., the anticancer potential of naphthoquinones either in combination with other treatment approaches or on their own is being more and more realized. The molecular mechanisms of naphthoquinones in cells mainly fall into two categories as inducing oxidant stress by ROS (reactive oxygen species) generation and directly interacting with traditional therapeutic targets in a non-oxidant mechanism. Based on this knowledge, optimized agents with naphthoquinones scaffold have been acquired and further tested. Hereby, we summarize the explored biological mechanisms of naphthoquinones in cells and review the application perspective of promising naphthoquinones in cancer therapies.

Design and characterization of α-lipoic acyl shikonin ester twin drugs as tubulin and PDK1 dual inhibitors.

Shikonin exhibits powerful anticancer activities for various cancer cells, but its poor solubility and strong toxicity hinder its development as clinical anticancer agent. We previously confirmed that shikonin and its derivatives can disturb mitosis through targeting tubulin. In this study, α-lipoic acid, the naturally-occurring co-factor of pyruvate dehydrogenase (PDH), was introduced into shikonin to design the twin drugs against both mitosis (tubulin) and glycolysis (PDK). 18 kinds of α-lipoic acid shikonin ester derivatives were achieved through three rounds of screening process performed by computer assistant drug design method, being designated as the outstanding compounds. Among them, 1c displayed the most potent cytotoxicity towards cervical cancer cells (HeLa) with an IC50 value of 3.14 ± 0.58 µM and inhibited xenotransplanted tumor growth in a dose-dependent manner. Further pharmacologic study demonstrated that 1c can cause cell cycle arrest in G2/M phase as tubulin polymerization inhibitor. Moreover, it also showed good PDK1 inhibitory activity, promoting PDH activity and forced HeLa cells to process more aerobic metabolism to undergo cell apoptosis. We reported here the first dual inhibitors of tubulin and PDK1 based on shikonin. It may form a basis for shikonin optimization through twin drug design framework for the discovery of new and potent shikonin derivatives in the study of targeted cancer therapy.

Effects of an EPSPS-transgenic soybean line ZUTS31 on root-associated bacterial communities during field growth.

The increased worldwide commercial cultivation of transgenic crops during the past 20 years is accompanied with potential effects on the soil microbial communities, because many rhizosphere and endosphere bacteria play important roles in promoting plant health and growth. Previous studies reported that transgenic plants exert differential effects on soil microbial communities, especially rhizobacteria. Thus, this study compared the soybean root-associated bacterial communities between a 5-enolpyruvylshikimate-3-phosphate synthase -transgenic soybean line (ZUTS31 or simply Z31) and its recipient cultivar (Huachun3 or simply HC3) at the vegetative, flowering, and seed-filling stages. High-throughput sequencing of 16S rRNA gene (16S rDNA) V4 hypervariable region amplicons via Illumina MiSeq and real-time quantitative PCR (qPCR) were performed. Our results revealed no significant differences in the overall alpha diversity of root-associated bacterial communities at the three developmental stages and in the beta diversity of root-associated bacterial communities at the flowering stage between Z31 and HC3 under field growth. However, significant differences in the beta diversity of rhizosphere bacterial communities were found at the vegetative and seed-filling stages between the two groups. Furthermore, the results of next generation sequencing and qPCR showed that the relative abundances of root-associated main nitrogen-fixing bacterial genera, especially Bradyrhizobium in the roots, evidently changed from the flowering stage to the seed-filling stage. In conclusion, Z31 exerts transitory effects on the taxonomic diversity of rhizosphere bacterial communities at the vegetative and seed-filling stages compared to the control under field conditions. In addition, soybean developmental change evidently influences the main symbiotic nitrogen-fixing bacterial genera in the roots from the flowering stage to the seed-filling stage.

Identification of Major Rhizobacterial Taxa Affected by a Glyphosate-Tolerant Soybean Line via Shotgun Metagenomic Approach.

The worldwide commercial cultivation of transgenic crops, including glyphosate-tolerant (GT) soybeans, has increased widely during the past 20 years. However, it is accompanied with a growing concern about potential effects of transgenic crops on the soil microbial communities, especially on rhizosphere bacterial communities. Our previous study found that the GT soybean line NZL06-698 (N698) significantly affected rhizosphere bacteria, including some unidentified taxa, through 16S rRNA gene (16S rDNA) V4 region amplicon deep sequencing via Illumina MiSeq. In this study, we performed 16S rDNA V5-V7 region amplicon deep sequencing via Illumina MiSeq and shotgun metagenomic approaches to identify those major taxa. Results of these processes revealed that the species richness and evenness increased in the rhizosphere bacterial communities of N698, the beta diversity of the rhizosphere bacterial communities of N698 was affected, and that certain dominant bacterial phyla and genera were related to N698 compared with its control cultivar Mengdou12. Consistent with our previous findings, this study showed that N698 affects the rhizosphere bacterial communities. In specific, N698 negatively affects Rahnella, Janthinobacterium, Stenotrophomonas, Sphingomonas and Luteibacter while positively affecting Arthrobacter, Bradyrhizobium, Ramlibacter and Nitrospira.

Transcriptome analysis explores genes related to shikonin biosynthesis in Lithospermeae plants and provides insights into Boraginales' evolutionary history.

Shikonin and its derivatives extracted from Lithospermeae plants' red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in "shikonin downstream biosynthesis" and "methyl jasmonate biosynthesis" were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.

Novel mechanisms for organic acid-mediated aluminium tolerance in roots and leaves of two contrasting soybean genotypes.

Aluminium (Al) toxicity is one of the most important limiting factors for crop yield in acidic soils. However, the mechanisms that confer Al tolerance still remain largely unknown. To understand the molecular mechanism that confers different tolerance to Al, we performed global transcriptome analysis to the roots and leaves of two contrasting soybean genotypes, BX10 (Al-tolerant) and BD2 (Al-sensitive) under 0 and 50 µM Al3+ treatments, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the expression levels of the genes involved in lipid/carbohydrate metabolism and jasmonic acid (JA)-mediated signalling pathway were highly induced in the roots and leaves of both soybean genotypes. The gene encoding enzymes, including pyruvate kinase, phosphoenolpyruvate carboxylase, ATP-citrate lyase and glutamate-oxaloacetate transaminase 2, associated with organic acid metabolism were differentially expressed in the BX10 roots. In addition, the genes involved in citrate transport were differentially expressed. Among these genes, FRD3b was down-regulated only in BD2, whereas the other two multidrug and toxic compound extrusion genes were up-regulated in both soybean genotypes. These findings confirmed that BX10 roots secreted more citrate than BD2 to withstand Al stress. The gene encoding enzymes or regulators, such as lipoxygenase, 12-oxophytodienoate reductase, acyl-CoA oxidase and jasmonate ZIM-domain proteins, involved in JA biosynthesis and signalling were preferentially induced in BD2 leaves. This finding suggests that the JA defence response was activated, possibly weakening the growth of aerial parts because of excessive resource consumption and ATP biosynthesis deficiency. Our results suggest that the Al sensitivity in some soybean varieties could be attributed to the low level of citrate metabolism and exudation in the roots and the high level of JA-mediated defence response in the leaves.

Identification of New Shikonin Derivatives as Antitumor Agents Targeting STAT3 SH2 Domain.

Signal transducer and activator of transcription 3 (STAT3) is hyper-activated in diversiform human tumors and has been validated as an attractive therapeutic target. Current research showed that a natural product, shikonin, along with its synthetic analogues, is able to inhibit the activity of STAT3 potently. The potential space of shikonin in developing novel anti-cancer agents encouraged us to carry out the investigation of the probable binding mode with STAT3. From this foundation, we have designed new types of STAT3 SH2 inhibitors. Combined simulations were performed to filter for the lead compound, which was then substituted, synthesized and evaluated by a variety of bioassays. Among the entities, PMM-172 exhibited the best anti-proliferative activity against MDA-MB-231 cells with IC50 value 1.98 ± 0.49 µM. Besides, it was identified to decrease luciferase activity, induce cell apoptosis and reduce mitochondrial transmembrane potential in MDA-MB-231 cells. Also, PMM-172 inhibited constitutive/inducible STAT3 activation without affecting STAT1 and STAT5 in MDA-MB-231 cells, and had no effect in non-tumorigenic MCF-10A cells. Moreover, PMM-172 suppressed STAT3 nuclear localization and STAT3 downstream target genes expression. Overall, these results indicate that the antitumor activity of PMM-172 is at least partially due to inhibition of STAT3 in breast cancer cells.

Impact of a Glyphosate-Tolerant Soybean Line on the Rhizobacteria, Revealed by Illumina MiSeq.

The global commercial cultivation of transgenic crops, including glyphosate-tolerant soybean, has increased widely in recent decades with potential impact on the environment. The bulk of previous studies showed different results on the effects of the release of transgenic plants on the soil microbial community, especially rhizosphere bacteria. In this study, comparative analyses of the bacterial communities in the rhizosphere soils and surrounding soils were performed between the glyphosate-tolerant soybean line NZL06-698 (or simply N698), containing a glyphosate-insensitive EPSPS gene, and its control cultivar Mengdou12 (or simply MD12), by a 16S ribosomal RNA gene (16S rDNA) amplicon sequencing-based Illumina MiSeq platform. No statistically significant difference was found in the overall alpha diversity of the rhizosphere bacterial communities, although the species richness and evenness of the bacteria increased in the rhizosphere of N698 compared with that of MD12. Some influence on phylogenetic diversity of the rhizosphere bacterial communities was found between N698 and MD12 by beta diversity analysis based on weighted UniFrac distance. Furthermore, the relative abundances of part rhizosphere bacterial phyla and genera, which included some nitrogen-fixing bacteria, were significantly different between N698 and MD12. Our present results indicate some impact of the glyphosate-tolerant soybean line N698 on the phylogenetic diversity of rhizosphere bacterial communities together with a significant difference in the relative abundances of part rhizosphere bacteria at different classification levels as compared with its control cultivar MD12, when a comparative analysis of surrounding soils between N698 and MD12 was used as a systematic contrast study.

Identification of new shikonin derivatives as STAT3 inhibitors.

The signal transducer and activator of transcription 3 is a constitutively activated oncogenic protein in various human tumors and represents a valid target for anticancer drug design. In this study, we have achieved a new type of STAT3 inhibitors based on structural modifications on shikonin scaffold, guided by computational modelling. By tests, PMMB-187 exhibited a more outstanding profile than shikonin on a small panel of human breast cancer cells, especially for the MDA-MB-231 cells. For the cellular mechanisms research, PMMB-187 was found to induce cell apoptosis in MDA-MB-231 cells, associated with the reduction of mitochondrial membrane potential, production of ROS and alteration of the levels of apoptosis-related proteins. Furthermore, PMMB-187 inhibited constitutive/inducible STAT3 activation, transcriptional activity, nuclear translocation and downstream target genes expression in STAT3-dependent breast cancer cells MDA-MB-231. Besides, no obvious inhibitory effect on activation of STAT1 and STAT5 was observed with PMMB-187 treatment. Most notably, the in vivo studies further revealed that PMMB-187 could dramatically suppress the MDA-MB-231 cells xenografted tumor growth. The in vitro and in vivo results collectively suggest that PMMB-187 may serve as a promising lead compound for the further development of potential therapeutic anti-neoplastic agents.

Comparison of miRNAs and Their Targets in Seed Development between Two Maize Inbred Lines by High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) play an important role in plant growth, development, and response to environment. For identifying and comparing miRNAs and their targets in seed development between two maize inbred lines (i.e. PH6WC and PH4CV), two sRNAs and two degradome libraries were constructed. Through high-throughput sequencing and miRNA identification, 55 conserved and 24 novel unique miRNA sequences were identified in two sRNA libraries; moreover, through degradome sequencing and analysis, 137 target transcripts corresponding to 38 unique miRNA sequences were identified in two degradome libraries. Subsequently, 16 significantly differentially expressed miRNA sequences were verified by qRT-PCR, in which 9 verified sequences obviously target 30 transcripts mainly involved with regulation in flowering and development in embryo. Therefore, the results suggested that some miRNAs (e.g. miR156, miR171, miR396 and miR444) related reproductive development might differentially express in seed development between the PH6WC and PH4CV maize inbred lines in this present study.

The LIKE SEX FOUR2 regulates root development by modulating reactive oxygen species homeostasis in Arabidopsis.

Maintaining reactive oxygen species (ROS) homeostasis plays a central role in plants, and is also critical for plant root development. Threshold levels of ROS act as signals for elongation and differentiation of root cells. The protein phosphatase LIKE SEX FOUR2 (LSF2) has been reported to regulate starch metabolism in Arabidopsis, but little is known about the mechanism how LSF2 affect ROS homeostasis. Here, we identified that LSF2 function as a component modulating ROS homeostasis in response to oxidative stress and, thus regulate root development. Compared with wild type Arabidopsis, lsf2-1 mutant exhibited reduced rates of superoxide generation and higher levels of hydrogen peroxide upon oxidative stress treatments. The activities of several antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase, were also affected in lsf2-1 mutant under these oxidative stress conditions. Consequently, lsf2-1 mutant exhibited the reduced root growth but less inhibition of root hair formation compared to wild type Arabidopsis plants. Importantly, protein phosphatase LSF2 interacted with mitogen-activated protein kinase 8 (MPK8), a known component of ROS homeostasis pathways in the cytoplasm. These findings indicated the novel function of LSF2 that controls ROS homeostasis to regulate root development.

Preparation, cellular uptake and angiogenic suppression of shikonin-containing liposomes in vitro and in vivo.

Shikonin has anticancer activity, but it has not yet been applied into clinical use. In the present study, shikonin was prepared using liposomes. We aimed to examine several aspects of sh-L (shikonin-containing liposomes): preparation, angiogenic suppression and cellular uptake through self-fluorescence. Sh-L were prepared using soybean phospholipid and cholesterol to form the membrane and shikonin was encapsulated into the phospholipid membrane. Three liposomes were prepared with shikonin. They had red fluorescence and were analysed using a flow cytometer. Angiogenic suppression of sh-L was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Transwell tests, chick CAM (chorioallantoic membrane) and Matrigel™ plug assay. MTT assay showed the median IC50 (inhibitory concentrations) as follows: shikonin, sh-L1 and sh-L2 were 4.99±0.23, 5.81±0.57 and 7.17±0.69 µM, respectively. The inhibition rates of migration were 53.58±7.05, 46.56±4.36 and 41.19±3.59% for 3.15 µM shikonin, sh-L1 and sh-L2, respectively. The results of CAM and Matrigel plug assay demonstrated that shikonin and sh-L can decrease neovascularization. Effect of shikonin was more obvious than sh-L at the same concentration. The results showed that sh-L decreased the toxicity, the rate of inhibition of migration and angiogenic suppression. The cellular uptake of the sh-L could be pictured because of the self-fluorescence. The self-fluorescence will be useful for conducting further research. Sh-L might be an excellent preparation for future clinical application to cancer patients.