Rong-Wu Yang

LeERF-1, a novel AP2/ERF family gene within the B3 subcluster, is down-regulated by light signals in Lithospermum erythrorhizon.

We previously showed that ethylene might be involved in the process of shikonin biosynthesis regulated by light signals. Here, we cloned a full-length cDNA of LeERF-1, a putative ethylene response factor gene, from Lithospermum erythrorhizon using the RACE (rapid amplification of cDNA ends) method. Phylogenetic analysis revealed that LeERF-1 was classified in the B3 subfamily, together with ERF1 and ORA59 of Arabidopsis. Heterologous expression of LeERF-1 in Arabidopsis showed that LeERF-1:eGFP fusion protein was precisely localised to the nucleus, implying that it might function as a transcription factor. Detailed expression analysis with real-time PCR showed that LeERF-1 was significantly down-regulated by white, blue and red light, although the inhibitory effect of red light was relatively weak compared to other light conditions. Tissue-specific expression analysis also indicated that LeERF-1 was dominantly expressed in the roots, which grow in soil in darkness. These patterns are all consistent with the effects of different light signals on regulating formation of shikonin and its derivatives, indicating that LeERF-1 might be a crucial positive regulator, like other B3 subfamily proteins (such as ORCA3 and ORA59), in regulating biosynthesis of secondary metabolites.

Synthesis and Biological Evaluation of Heterocyclic Carboxylic Acyl Shikonin Derivatives

A series of shikonin derivatives (1–13) that were acylated selectively by various thiophene or indol carboxylic acids at the side chain of shikonin were synthesized, and their biological activities were also evaluated as potential tubulin inhibitors. Among them, compound 3 ((R)‐1‐(5,8‐dihydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐4‐methylpent‐3‐enyl 3‐(1H‐indol‐3‐yl)propanoate) and compound 8 ((R)‐1‐(5,8‐dihydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐4‐methylpent‐3‐enyl 2‐(thiophen‐3‐yl)acetate) exhibited good antiproliferative activity of A875 (IC50 = 0.005 ± 0.001 μm, 0.009 ± 0.002 μm) and HeLa (IC50 = 11.84 ± 0.64 μm, 4.62 ± 0.31 μm) cancer cell lines in vitro, respectively. Shikonin (IC50 = 0.46 ± 0.002 μm, 4.80 ± 0.48 μm) and colchicine (IC50 = 0.75 ± 0.05 μm, 17.79 ± 0.76 μm) were used as references. Meanwhile, they also showed the most potent growth inhibitory activity against tubulin (IC50 of 3.96 ± 0.13 μm and 3.05 ± 0.30 μm, respectively), which were compared with shikonin (IC50 = 15.20 ± 0.25 μm) and colchicine (IC50 = 3.50 ± 0.35 μm). Furthermore, from the results of flow cytometer, we found compound 3 can really inhibit HeLa cell proliferation and has low cell toxicity. Based on the preliminary results, compound 3 with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-norcantharidin hybrid drugs.

In this study, we designed and synthesized eighteen podophyllotoxin-norcantharidin hybrid drugs which could exhibit more potent anti-cancer activity than the parent drugs. Through the anti-proliferation assay, the most potent anti-cancer agent was screened out, namely Q9 (IC50=0.88±0.18μM against MCF-7 cell line), and it showed lower cytotoxicity against non-cancer cells, human embryonic kidney cells (293T) (IC50=54.38±3.78μM). Additionally, based on the flow cytometry analysis result, it can cause a remarkable cell cycle arrest at G2/M phase and induce apoptosis in MCF-7 cells more significantly than podophyllotoxin or norcantharidin per se. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while a protein required for mitotic initiation, Cyclin B1 was down regulated. Furthermore, according to the confocal microscopy observation results, it was shown that Q9 was a potent tubulin polymerization inhibitor and the effect is comparable to that of colchicine. For further investigation on the aforementioned mechanisms, we performed western blot experiments, thus finding the increase of the cleavage of PARP. Consistent with these new findings, molecular docking observations suggested that compound Q9 could be developed as a potential anticancer agent.

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.

Design, Synthesis, and Biological Evaluation of Chalcone-Containing Shikonin Derivatives as Inhibitors of Tubulin Polymerization.

The biological importance of microtubules in mitosis makes them an interesting target for the development of anticancer agents. In this study, a series of novel chalcone‐containing shikonin derivatives was designed, synthesized, and evaluated for biological activities. Among them, derivative PMMB‐259 [(R)‐1‐(5,8‐dihydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐4‐methylpent‐3‐en‐1‐yl (E)‐2‐(4‐(3‐oxo‐3‐(3‐(trifluoromethoxy)phenyl)prop‐1‐en‐1‐yl)phenoxy)acetate] was identified as a potent inhibitor of tubulin polymerization. Further investigation confirmed that PMMB‐259 can induce MCF‐7 cell apoptosis, reduce the mitochondrial transmembrane potential, and arrest the cell cycle at the G2/M phase. Moreover, the morphological variation of treated cells was visualized by confocal microscopy. The results, along with docking simulations, further indicated that PMMB‐259 can bind well to tubulin at the colchicine site. Overall, these studies may provide a new molecular scaffold for the further development of antitumor agents that target tubulin.

Identification of New Shikonin Derivatives as STAT3 Inhibitors.

Graphical abstract Identification of new potent STAT3 inhibitors based on the interaction model of natural product shikonin (A), the lead compound (B), and its optimization (C). Figure. No Caption available. ABSTRACT 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.

Transgenic studies reveal the positive role of LeEIL-1 in regulating shikonin biosynthesis in Lithospermum erythrorhizon hairy roots

BackgroundThe phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation.ResultsThe mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production.ConclusionsThe results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

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 remain largely unknown. Based on the global transcriptome analysis of the roots and leaves of two contrasting soybean genotypes, BX10 (Al-tolerant) and BD2 (Al-sensitive) under 0 µM and 50 µM Al3+ treatments, our findings suggest that BX10 can resist Al by secreting additional citrate into the rhizosphere from the roots to chelate Al and by avoiding a JA-mediated defense response that allows resource allocation to maintain leaf growth.

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.