Gui-Hua Lu

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.

Synthesis and biological evaluation of novel shikonin ester derivatives as potential anti-cancer agents

Shikonin has previously been reported to function as a potent anti-cancer drug that induces cell apoptosis via diverse pathways. To optimize the effectiveness of its pro-apoptotic functions, shikonin was chosen as the best compound for obtaining shikonin ester derivatives (3a–3n) because it possesses an ester group on the side chain hydroxyl position, and the biological activity was evaluated as a potent anti-proliferating inhibitor for many cancer cell lines. Among these compounds, compound 3j exhibited better anti-cancer activities against human hepatocellular carcinoma cell line (HepG2) with an IC50 value of 0.759 μM which was better than that of shikonin which was 1.288 μM. The flow cytometry results showed that compound 3j inhibited the cell growth and caused the cell cycle to be arrested at the G2/M phase. Meanwhile, obvious apoptosis induced by 3j was observed using the Annexin V combined with propidium iodide assay, showing that 3j induced apoptosis of HepG2 cells in a dose and time dependent manner. To investigate the underlying mechanism in the process of apoptosis induced by 3j, the western blot technique was used and the cleavage of caspase-9 was observed but not that of caspase-8. Furthermore, the elevated expression of the p53 protein was positively correlated with the decreased Bcl-2 protein levels and increased Bax protein and the cytochrome C levels. This indicated that the mitochondrial apoptosis pathway directed by p53 responded in the process of apoptosis was induced by 3j. Based on these data, we conclude that compound 3j has the best anti-proliferating and pro-apoptotic effect among the 14 newly synthesized compounds, and it could be a potent candidate for cancer therapy.

Design, synthesis and mechanism of novel shikonin derivatives as potent anticancer agents

In this study, a series of novel shikonin derivatives (30–49) were designed and synthesized and their anti-proliferative activities were evaluated against five different cancer cell lines, including HeLa, HepG2, MCF-7, BGC and A549. Some of the compounds show strong anti-proliferative effects against HeLa, HepG2 and MCF-7 with IC50 values ranging from 1.26 to 18.50 μM and show lower side effects towards normal cell lines as compared to shikonin. Compared to other compounds and shikonin itself, compound 40 displayed much stronger anti-proliferative effects against various cancer cell lines. Furthermore, the flow cytometry results demonstrated that compound 40 could obviously induce apoptosis in a dose- and time-dependent manner and also cause cell cycle arrest at the G2/M phase. For further investigation of the aforementioned mechanisms, we performed Western blot experiments and found that the cleavage of PARP and upstream caspase-3 increased; moreover, caspase-9 was activated by cleavage but not caspase-8. These aforementioned results also indicate that compound 40 could induce caspase-9 involved apoptosis and G2/M phase cell cycle arrest via the P21, p-CDC2 (Tyr15) pathway independent of P53.

Design and synthesis of piperazine acetate podophyllotoxin ester derivatives targeting tubulin depolymerization as new anticancer agents

In this paper, a series of podophyllotoxin piperazine acetate ester derivatives were synthesized and investigated due to their antiproliferation activity on different human cancer cell lines. Among the congeners, C5 manifested prominent cytotoxicity towards the cancer cells, without causing damage on the non-cancer cells through inhibiting tubulin assembly and having high selectively causing damage on the human breast (MCF-7) cell line (IC50=2.78±0.15μM). Treatments of MCF-7 cells with C5 resulted in cell cycle arrest in G2/M phase and microtubule network disruption. Moreover, regarding the expression of cell cycle relative proteins CDK1, a protein required for mitotic initiation was up-regulated. Besides, Cyclin A, Cyclin B1 and Cyclin D1 proteins were down-regulated. Meanwhile, it seems that the effect of C5 on MCF-7 cells apoptosis inducing was observed to be not obvious enough. In addition, docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

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.

Overexpression of LeMYB1 enhances shikonin formation by up-regulating key shikonin biosynthesis-related genes in Lithospermum erythrorhizon

We previously reported that LeMYB1 might be a crucial transcription factor in regulating shikonin formation in Lithospermum erythrorhizon. In this study, by overexpressing LeMYB1 under the control of CaMV35S promoter in L. erythrorhizon hairy roots, we further clarified the role of LeMYB1 in the shikonin formation and its regulation. The LeMYB1-overexpressing transgenic hairy roots were successfully induced by infecting seedling nodes with Agrobacterium rhizogenes strain ATCC15834 that carried the pBI121-LeMYB1 vector. The LeMYB1 transcripts were significantly up-regulated in the transgenic hairy root lines compared with the wild type lines, and the total content of shikonin and its derivatives was dramatically enhanced by the LeMYB1 overexpression. Real-time PCR results reveal that the enhanced shikonin biosynthesis in the overexpressing lines were mainly caused by a highly up-regulated expression of genes coding key enzymes (PAL, HMGR, and PGT) and key regulators (LeDI-2 and LePS-2) involved in the shikonin biosynthesis. Overall, our results suggest that LeMYB1 plays a positive role in regulating the shikonin biosynthesis in L. erythrorhizon.

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.

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.