Hong-Wei Han

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 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.

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.

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.

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 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.

The evaluation of potent antitumor activities of shikonin coumarin-carboxylic acid, PMMB232 through HIF-1α-mediated apoptosis

In current study, a series of shikonin derivatives were synthesized and its anticancer activity was evaluated. As a result, PMMB232 showed the best antiproliferation activity with an IC50 value of 3.25±0.35μM. Further, treatment of HeLa cells with a variety of concentrations of target drug resulted in dose-dependent event marked by apoptosis. Whats more, the mitochondrial potential (Δym) analysis was consistent with the apoptosis result. In addition, PARP was involved in the progress of apoptosis revealed by western blotting. To identify the detailed role and mechanism of PMMB232 in the progression of human cervical cancer, we detected the expression of HIF-1α and E-cadherin in HeLa cells. Results showed that expression of HIF-1α was downregulated, while E-cadherin protein was upregulated. Meanwhile, glycolysis related protein PDK1 was decreased in HeLa cells. Conversely, the expression of PDH-E1α was upregulated. Docking simulation results further indicate that PMMB232 could be well bound to HIF-1α. Taken together, our data indicate that compound PMMB232 could be developed as a potential anticancer agent.

Synthesis, characterization and biological evaluation of formononetin derivatives as novel EGFR inhibitors via inhibiting growth, migration and inducing apoptosis in breast cancer cell line

Over the past few decades, the human epidermal growth factor receptor (EGFR) has been established as an attractive target for non-small cell lung cancer (NSCLC) therapy. Nevertheless, the approved EGFR inhibitors, gefitinib or erlotinib have shown minimum clinical activity to breast cancer patients, who also highly expressed EGFR. In this study, we designed and synthesized a series of novel formononetin derivatives by reference to the binding mode of lapatinib to EGFR. In vitro EGFR and cell growth inhibition assay demonstrated that compound 4v exhibited the most potent anti-EGFR (IC50 = 14.5 nM) and anti-proliferation activity (IC50 = 5.44 ± 1.28 μM) against MDA-MB-231 cell line, which was comparable to that of lapatinib (EGFR, IC50 = 5.6 nM; MDA-MB-231, IC50 = 2.48 ± 1.04 μM). Further biological experiment results demonstrated that 4v could effectively induce apoptosis, inhibit proliferation and migration in MDA-MB-231 cells through targeting EGFR and then blocking the downstream signaling pathways, EGFR/PI3K/Akt/Bad, EGFR/ERK and EGFR/PI3K/Akt/β-catenin, respectively. However, it had no significant influence on cell cycle distribution and the related proteins (Cyclin A, Cyclin D1, CDK4) expression. In vivo anti-tumor results also preliminarily confirmed the effectiveness of 4v in tumor chemotherapy in mice and indicated its potential as a new EGFR inhibitor in the treatment of MDA-MB-231 malignant tumor.

Design, synthesis and anti-cancer evaluation of novel podophyllotoxin derivatives as potent tubulin-targeting agents

A series of podophyllotoxin derivatives (M1–M16) that were selectively acylated by various phenoxy acids at the C-4 position of podophyllotoxin were synthesized, and their biological activities were also evaluated. Among them, compound M4 showed the most potent anti-cancer activity against HeLa cells with an IC50 value of 1.64 ± 0.41 μM. Additionally, flow cytometry analysis results indicated that it could cause a remarkable cell cycle arrest at G2/M phase, but the effect on apoptosis inducing was not significant. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while cyclin B1 and Cdc25C, two proteins required for mitotic initiation were down regulated. Furthermore, the confocal assay and extracellular polymerized tubulin expression analysis also demonstrated that M4 was a potent tubulin polymerization inhibitor and the effect was comparable to that of colchicine. Finally, docking simulation results showed that M4 could nicely bind to the colchicine binding site of tubulin which further comfirmed the tubulin inhibiton activity of M4.

Novel Podophyllotoxin Derivatives as Potential Tubulin Inhibitors: Design, Synthesis and Anti-proliferative Activity Evaluation

A number of podophyllotoxin derivatives (3A–3J) had been designed and synthesized, and their biological activities were evaluated in this study. Moreover, the antiproliferation activities of these compounds against four human cancer cell lines (HepG2, HeLa, A549, and MCF‐7) were also tested. The results indicated that the most promising compound 3D displayed potent inhibitory activity over the four human cancer cell lines and was further demonstrated to have potent tubulin polymerization inhibitory effects without damaging the non‐cancer cells. Additionally, 3D was verified to effectively interfere with tubulin and could prevent the mitosis of cancer cells, leading to cell cycle arrest and eventually inducing apoptosis in a dose‐ and time‐dependent manner. Moreover, the Western blotting and siRNA results showed that Bcl‐2 was downregulated in HepG2 cells treated with 3D. Finally, the molecular docking simulation results revealed that 3D could fit well in the colchicine‐binding pocket. Taken together, this study has provided certain novel antitubulin agents for possible cancer chemotherapy.