Hong-Yan Lin

Novel Shikonin Derivatives Targeting Tubulin as Anticancer Agents

In this study, we report the identification of a new shikonin‐phenoxyacetic acid derivative, as an inhibitor of tubulin. A series of compounds were prepared; among them, compound 16 [(R) ‐1 ‐ (5, 8‐ dihydroxy‐1, 4‐ dioxo‐1, 4‐ dihydronaphthalen‐2‐yl)‐4‐methylpent‐3‐enyl 2‐ (4‐ phenoxyphenyl) acetate] potently inhibited the function of microtubules, inducing cell growth inhibition, apoptosis of cancer cell lines in a concentration and time‐dependent manner. Molecular docking involving 16 at the vinblastine binding site of tubulin indicated that a phenoxy moiety interacted with tubulin via hydrogen bonding with asparaginate (Asn) and tyrosine (Tyr). Analysis of microtubules with confocal microscopy demonstrated that 16 altered the microtubule architecture and exhibited a significant reduction in microtubule density. Cell cycle assay further proved that HepG2 cells were blocked in G2/M phase. Our study provides a new, promising compound for the development of tubulin inhibitors by proposing a new target for the anticancer activity of shikonin.

Design, Synthesis and Biological Evaluation of Cinnamic Acyl Shikonin Derivatives

Inducing apoptosis is an important and promising therapeutic approach to overcome cancer. Here, we described a series of novel synthesized compounds, cinnamic acyl shikonin derivatives (1b–19b), which were synthesized starting from shikonin and cinnamic acids, which exhibit anticancer activity via inducing apoptosis in vitro. Our flow cytometry results showed that compound 8b((E)‐1‐(5,8‐dihydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐4‐methylpent ‐3‐enyl‐3‐(3‐(trifluoromethyl) phenyl)acrylate) (IC50 = 0.69, 0.65, 1.62 μm for human SW872‐s, A875 and A549 cell lines, respectively) exhibited conspicuous anticancer activities and has low cell toxicity in vitro. Therefore, we considered that compound 8b is potentially to be a candidate of anticancer agent. The proliferation inhibitory effect of compound 8b was associated with its apoptosis‐inducing effect by activating caspase‐3, caspase‐7, caspase‐9, and PARP. When the level of cleaved caspase‐3, cleaved caspase‐7, cleaved caspase‐9, and cleaved PARP are rise, apoptosis of cancer cells will be induced.

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 biological evaluation of shikonin thio-glycoside derivatives: new anti-tubulin agents

A novel series of acetyl-β-D-thio-glycoside modified shikonin derivatives were designed and synthesized and investigated for inhibition of cell proliferation against MG63, MCF-7, B16-F10, HepG2, MDA-231, L02, VERO and MCF-10A cell lines. The biological study showed that most single, di- and tri-substituted shikonin derivatives exhibited better anti-proliferative activities against the five cancer cell lines but lower cytotoxic activity against normal cells than shikonin itself. Notably, compared to shikonin, IIb displayed much stronger anti-proliferative effect among them. Furthermore, the inhibition of tubulin polymerization results indicated that IIb showed the most potent anti-tubulin activity (IC50 = 4.67 ± 0.433 μM), which was compared with shikonin (IC50 = 16.8 ± 0.625 μM) and colchicine (IC50 = 3.83 ± 0.424 μM). Docking simulation, confocal microscopy and western bolt results further confirmed that IIb can cause cell arrest in G2/M phase and induce cell apoptosis via binding to the active site of tubulin and inhibiting tubulin polymerization.

Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents through microtubule stabilization

The high incidence of cancer and the side effects of traditional anticancer drugs motivate the search for new and more effective anticancer drugs. In this study, we synthesized 17 kinds of aryl dihydrothiazol acyl shikonin ester derivatives and evaluated their anticancer activity through MTT assay. Among them, C13 showed better antiproliferation activity with IC50=3.14 ± 0.21 μM against HeLa cells than shikonin (IC50=5.75 ± 0.47 μM). We then performed PI staining assay, cell cycle distribution, and cell apoptosis analysis for C13 and found that it can cause cell arrest in G2/M phase, which leads to cell apoptosis. This derivative can also reduce the adhesive ability of HeLa cells. Docking simulation and confocal microscopy assay results further indicated that C13 could bind well to the tubulin at paclitaxel binding site, leading to tubulin polymerization and mitotic disruption.

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.

Semi-synthesis and anti-lung cancer activity evaluation of aryl dihydrothiazol acyl podophyllotoxin ester derivatives

Lung cancer is the leading cause of cancer death worldwide, making it one of the biggest concerns for chemoprevention. In this study, we obtained seventeen potent anticancer agents through semi-synthesis based on a natural product, podophyllotoxin. Despite prior studies of podophyllotoxin derivatives being focussed on DNA-topoisomerase II, we now turn our attention to their effect on tubulin. The MTT assay screened out the most potent anticancer agent, S12 (IC50 = 0.18 μM against A549 cell line), and it showed lower cytotoxicity against normal cells. Next, the flow cytometry analysis result demonstrated that it can cause a remarkable cell cycle arrest at G2/M phase but the effect on apoptosis is not very significant. In addition, docking simulation results showed that S12 can nicely bound to the colchicine binding site of tubulin. Furthermore, we confirmed that S12 can really inhibit tubulin polymerization through confocal microscopy and protein expression determination assay.

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.

Synthesis of novel aryl dithian valeryl podophyllotoxin ester derivatives as potential antitubulin agents

Microtubules are among the most successful targets for anticancer therapy. In this study, we described the synthesis routes of the lipoyl podophyllotoxin ester derivatives and found that they can selectively inhibit the proliferation of cancer cells without damaging the non-cancer cells. Among them, L4 showed the best antiproliferation activity with IC50 = 2.68 μM against A549 cells. This effect of L4 was similar to that of CA-4 (IC50 = 2.78 μM), a typical microtubule inhibitor, but better than podophyllotoxin (IC50 = 6.57 μM) itself. Furthermore, cell cycle analysis revealed that L4 can remarkably cause cell cycle arrest in the G2/M phase in a time- and dose-dependent manner. But the effect of L4 on apoptosis inducing was not apparent enough. Moreover, confocal microscopy and western blot analysis results indicated that L4 can perturb microtubule polymerization, thus causing tumor growth inhibition.

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.