Design, synthesis and biological evaluation of colchicine glycoconjugates as tubulin polymerization inhibitors.

A series of new colchicine glycoconjugates as tubulin polymerization inhibitors were designed by targeting strategy based on Warburg effect. All of the colchicine glycoconjugates were synthesized and then evaluated for their antiproliferative activities against three human cancer lines HT-29, MCF-7 and Hep-3B. Among them, 1e exhibited greater than 10 times selectivity between GLUT1 highly expressed cells (HT-29 and MCF-7) and GLUT1 lowly expressed cells (Hep-3B), and also showed lower cytotoxicity against HUVECs compared with colchicine. Moreover, 1e significantly inhibited tubulin polymerization and disrupted microtubule networks. GLUT1 inhibitor-dependent cytotoxicity assay demonstrated that the uptake of 1e was regulated via GLUT1. Molecular docking studies showed that 1e could be a substrate of GLUT1 and bind to the colchicine site of tubulin.

The anti-MDR efficacy of YAN against A549/Taxol cells is associated with its inhibition on glycolysis and is further enhanced by 2-deoxy-d-glucose.

Aerobic glycolysis is a hallmark of malignant tumor. Here, the hyperactive glycolysis in multidrug-resistant A549/Taxol cells was demonstrated to be essential for maintaining the vigorous cell viability and drug resistance. 5-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-3-amine (YAN), a newly synthesized tubulin inhibitor, could not only inhibit the glycolysis in A549 and A549/Taxol cells through down-regulating the glycolysis-related proteins, but also disrupt the mitochondrial localization of hexokinase-2 (HK-2) which is related with the apoptosis resistance. The effects of YAN above were relevant to the down-regulation of PI3K-Akt-c-Myc/HIF-1α pathway. Moreover, YAN induced the reactive oxygen species generation in A549 and A549/Taxol cells, which only mediated the apoptosis in A549 cells. We also showed that 2-DG, the glycolysis inhibitor, synergistically enhanced YAN-triggered apoptosis in A549/Taxol cells via further suppressing glycolysis and reducing mitotic slippage. Collectively, we illustrate the inhibition effect of YAN on the glycolysis in A549 and A549/Taxol cells, and provide a fresh insight into the mechanism for the development of YAN as a candidate for multidrug resistant cancer treatment. The finding that 2-DG improved the anti-tumor efficacy of YAN against A549/Taxol cells, offers a reference for solving mitotic slippage-mediated drug resistance.

EML4-ALK G1202R mutation induces EMT and confers resistance to ceritinib in NSCLC cells via activation of STAT3/Slug signaling.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

Design, synthesis and anticancer activity of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as microtubule-destabilizing agents.

Hereby, we report our efforts on discovery and optimization of a new series of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-1,2,3-thiadiazoles as new microtubule-destabilizing agents along our previous study. Guided by docking model analysis, we introduced the 1,2,3-thiadiazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. Extensive structure modifications were performed to investigate the detailed structure and activity relationships (SARs). Some compounds exhibited potent antiproliferative activities against three human cancer cell lines (SGC-7901, A549 and HeLa). The compound 5m exhibited the highest potency against the three cancer cell lines. The tubulin polymerization experiments indicated that compound 5m effectively inhibited the tubulin polymerization, and immunostaining assay revealed that it significantly disrupted microtubule dynamics. Moreover, cell cycle studies revealed that compound 5m dramatically arrested cell cycle progression at G2/M phase.

YAN, a novel microtubule inhibitor, inhibits P-gp and MRP1 function and induces mitotic slippage followed by apoptosis in multidrug-resistant A549/Taxol cells.

Lung cancer is the most common cause of cancer-related death worldwide. The occurrence of multidrug resistance (MDR) affects the therapeutic efficacy of chemotherapeutics. Therefore, to develop new anticarcinogen which can overcome MDR is urgent. Here, the novel microtubule inhibitor 5-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-3-amine (YAN) exhibited strong cytotoxicity towards A549 and MDR-phenotype A549/Taxol cells. We demonstrated that YAN was a poor substrate of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) which were over-expressed in A549/Taxol cells, and YAN inhibited their expression and function. Moreover, YAN arrested cells at mitosis phase by inhibiting microtubule polymerization. Further, YAN induced caspase-dependent apoptosis in A549 cells via mitochondria-mediated intrinsic pathway. In contrast, the multinucleation of A549/Taxol cells after YAN-treatment indicated the occurrence of mitotic catastrophe, and the subsequent apoptosis was mediated by apoptosis-inducing factor (AIF) nuclear translocation instead of p53- and caspase-dependent manner. Moreover, the inhibitory effect of YAN on PI3K/Akt activity was involved in the regulation of P-gp, MRP1 and AIF in A549/Taxol cells. Taken together, our finding indicates that YAN is a novel microtubule inhibitor and overcomes MDR by suppressing P-gp and MRP1 function and inducing cell death independent of p53 and caspase in A549/Taxol cells. Therefore, YAN possesses great potential for future development into an effective anticarcinogen especially for drug-resistant cancer.

MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial-mesenchymal transition in A549/Taxol cells.

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.

3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1,2,4] triazolo [3,4-b][1,3,4] thiadiazine, a novel tubulin inhibitor, evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells.

Gastric cancer and cervical cancer are two major malignant tumors that threaten human health. The novel chemotherapeutic drugs are needed urgently to treat gastric cancer and cervical cancer with high anticancer activity and metabolic stability. Previously we have reported the synthesis, characterization and identification of a novel combretastatin A-4 analog, 3-(3-methoxyphenyl)-6-(3-amino-4- methoxyphenyl) -7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine (XSD-7). In this study, we sought to investigate its anticancer mechanisms in a human gastric cancer cell line (SGC-7901 cells) and human cervical carcinoma cell line (HeLa cells). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that XSD-7 induced cytotoxicity in SGC-7901 and HeLa cells with inhibitory concentration 50 values of 0.11 ± 0.03 and 0.12 ± 0.05 µM, respectively. Immunofluorescence studies proved that XSD-7 inhibited microtubule polymerization during cell division in SGC-7901 and HeLa cells. Then, these cells were arrested at G2/M cell cycle and subsequently progressed into apoptosis. In further study, mitochondrial membrane potential analysis and Western blot analysis demonstrated that XSD-7 treatment-induced SGC-7901 cell apoptosis via both the mitochondria-mediated pathway and the death receptor-mediated pathway. In contrast, XSD-7 induced apoptosis in HeLa cells mainly via the mitochondria-mediated pathway. Hence, our data indicate that XSD-7 exerted antiproliferative activity by disrupting microtubule dynamics, leading to cell cycle arrest, and eventually inducing cell apoptosis. XSD-7 with novel structure has the potential to be developed for therapeutic treatment of gastric cancer and cervical cancer.

Design, synthesis and biological evaluation of 3,4-diaryl-1,2,5-oxadiazole-2/5-oxides as highly potent inhibitors of tubulin polymerization.

Structure-activity relationships for rigid analogues of combretastatin A-4 (CA-4) were investigated, leading to the discovery of a series of 3,4-diaryl-1,2,5-oxadiazole-N-oxides. Among them, 7n' and 7n'' showed remarkable antiproliferative activities against three cancer cell lines in nanomolar concentrations. Interestingly, 7n' inhibited tubulin polymerization much more efficiently than CA-4. Cellular mechanism investigation elucidated 7n' disrupted the cellular microtubule structure, arrested cell cycle at G2/M phase and induces apoptosis. Molecular modeling study revealed 1,2,5-oxadiazole-N-oxide ring could increase a hydrogen bond interaction with the binding site. These results provide impetus and further guidance for the development of new CA-4 analogues.

Chemical composition and biological activities of Gerbera anandria.

Gerbera anandria (Compositae) was extracted with 75% ethanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. Column chromatography of the light petroleum fraction resulted in the isolation of methyl hexadecanoate, while the chloroform fraction afforded xanthotoxin, 2-hydroxy-6-methylbenzoic acid, 7-hydroxy-1(3H)-isobenzofuranone, a mixture of ß-sitosterol and stigmasterol, and 8-methoxysmyrindiol and the ethyl acetate fraction gave gerberinside, apigenin-7-O-ß-d-glucopyranoside and quercetin. A new coumarin, 8-methoxysmyrindiol, was found. The chemical structures of the isolated compounds were established by MS and NMR (HSQC, HMBC). Free radical scavenging and cytotoxic activities of crude extracts and 8-methoxysmyrindiol were further investigated. The ethyl acetate phase exerted the strongest DPPH free radical scavenging activity in comparison to the other fractions. The coumarin 8-methoxysmyrindiol demonstrated cytotoxicity against multiple human cancer cell lines, with the highest potency in HepG2 cells.