Marine Compound Exerts Antiaging Effect in Human Endothelial Progenitor Cells via Increasing Sirtuin1 Expression.

Aging is associated with an increased risk of cardiovascular disease. Previous studies have demonstrated that compound 3 (C3), a derivative of marine compound xyloallenoide A isolated from the mangrove fungus Xylaria sp. (no. 2508), exhibited strong angiogenic activities in zebrafish. In this study, we examined the effects of C3 on the senescence of endothelial progenitor cells isolated from human peripheral blood (hEPCs). The results showed that treatment with angiotensin II (AngII) for 24 h induced hEPC senescence, as demonstrated by increased SA-ß-galactosidase staining. Moreover, there is a significant decrease in telomerase activity and cellular viability in AngII-treated hEPCs. These changes in aging hEPCs were greatly recovered by C3 in a dose-dependent manner. Furthermore, C3 significantly restored the AngII-induced decrease of sirtuin type 1 (SIRT1) expression, a well-known antiaging protein. In addition, AngII increased AMP-activated protein kinase (AMPK) phosphorylation and reduced Akt phosphorylation in aging hEPCs, which were also reversed by C3. Importantly, the inhibition of C3 on hEPC senescence and AMPK/Akt dysregulation was significantly attenuated by the SIRT1-specific inhibitor nicotinoyl. These results indicated that C3 protects hEPC against AngII-induced senescence by increasing SIRT1 expression levels and balancing the AMPK/Akt signaling pathway. The inhibition of hEPCs senescence by C3 might protect EPCs against dysfunction induced by pathological factors in the elderly population. C3 may provide a novel drug candidate for the treatment of aging-related disorders.

Xyloketal B exerts antihypertensive effect in renovascular hypertensive rats via the NO-sGC-cGMP pathway and calcium signaling.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 µmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.

Marine derived xyloketal derivatives exhibit anti-stress and anti-ageing effects through HSF pathway in Caenorhabditis elegans.

Ageing is a complex but universal phenomenon that progressively challenges the homeostasis network and finally leads to the dysfunction of organisms and even death. Previous studies demonstrated that xyloketal B and its derivatives, a series of marine novel ketone compounds, possessed unique antioxidative effects on endothelial and neuronal oxidative injuries. In this study, we examined the effects of xyloketal derivatives on extending lifespan and healthspan of Caenorhabditis elegans. The results showed that most selected xyloketals could protect Caenorhabditis elegans against heat stress and extend the lifespan of worms. Compound 15, a benzo-1, 3-oxazine xyloketal derivative, possessed most potent effect in anti-heat stress assay and significantly attenuated ageing-related decrease of pumping and bending of the worms in healthspan assay. In addition, the beneficial effect of 15 was abolished in PS3551 worms, a strain that possesses non-functional heat shock transcription factor-1 (HSF-1). Furthermore, 15 increased the expression of heat shock protein 70 (HSP70), a downstream molecular chaperone of HSF-1. These results indicated that HSF-1 might contribute to the protective effect of this compound in Caenorhabditis elegans ageing. Molecular docking studies suggested that these xyloketal derivatives were bound to the DNA binding domain of HSF-1, promoted the conformation of HSF-1, thus strengthened the interaction between the HSF-1 and related DNA. ALA-67, ASN-74 and LYS-80 of binding region might be the key amino residues during the interaction. Finally, compound 15 could reduce the paralysis of the CL4176 worms, a transgenic strain expressing human Aß3-42 under a temperature-inducible system. Collectively, these data indicate that xyloketals have potential implications for further evaluation in anti-ageing studies.

Recent Advances in the Discovery and Development of Marine Natural Products with Cardiovascular Pharmacological Effects.

Numerous studies have indicated that marine natural products are one of the most important sources of the lead compounds in drug discovery for their unique structures, various bioactivities and less side effects. In this review, the marine natural products with cardiovascular pharmacological effects reported after 2000 will be presented. Their structural types, relevant biological activities, origin of isolation and information of strain species will be discussed in detail. Finally, by describing our studies as an example, we also discuss the chances and challenges for translating marine-derived compounds into preclinical or clinical trials.

Xyloketal B alleviates cerebral infarction and neurologic deficits in a mouse stroke model by suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. We previously demonstrated that pretreatment with Xyl-B exerted neuroprotective effects and attenuated hypoxic-ischemic brain injury in neonatal mice. In the present study we investigated the neuroprotective effects of pre- and post-treatment with Xyl-B in adult mice using a transient middle cerebral artery occlusion (tMCAO) model, and explored the underlying mechanisms. Adult male C57 mice were subjected to tMCAO surgery. For the pre-treatment, Xyl-B was given via multiple injections (12.5, 25, and 50 mg·kg-1·d-1, ip) 48 h, 24 h and 30 min before ischemia. For the post-treatment, a single dose of Xyl-B (50 mg/kg, ip) was injected at 0, 1 or 2 h after the onset of ischemia. The regional cerebral perfusion was monitored using a laser-Doppler flowmeter. TTC staining was performed to determine the brain infarction volume. We found that both pre-treatment with Xyl-B (50 mg/kg) and post-treatment with Xyl-B (50 mg/kg) significantly reduced the infarct volume, but had no significant hemodynamic effects. Treatment with Xyl-B also significantly alleviated the neurological deficits in tMCAO mice. Furthermore, treatment with Xyl-B significantly attenuated ROS overproduction in brain tissues; increased the MnSOD protein levels, suppressed TLR4, NF-κB and iNOS protein levels; and downregulated the mRNA levels of proinflammatory cytokines, including IL-1ß, TNF-α, IL-6 and IFN-γ. Moreover, Xyl-B also protected blood-brain barrier integrity in tMCAO mice. In conclusion, Xyl-B administered within 2 h after the onset of stroke effectively protects against focal cerebral ischemia; the underlying mechanism may be related to suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

TRC4, an improved triptolide derivative, specifically targets to truncated form of retinoid X receptor-alpha in cancer cells.

The nuclear retinoid X receptor-α (RXRα) plays critical roles in cell homeostasis and in many physiological processes mainly through its transcriptional function. However, an N-terminal truncated form of RXRα, tRXRα, was frequently described in various cancer cells and tumor tissues, thus representing a new promising drug target. We recently demonstrated that triptolide (TR01) could target to the oncogenic activity of tRXRα. To improve its tumor selectivity, we developed several TR01 derivatives by introducing different amine ester groups on C-14-hydroxyl site. Interestingly, C-14 modification could differently affect the expression of tRXRα without interfering the level of its full length RXRα. Among the derivatives, TRC4 could strongly reduce tRXRα expression, while TRC5-7 increased it. The capability of inhibiting tRXRα expression was shown to be closely associated with its inactivation of AKT and induction of apoptosis in various cancer cells. Conversely, treatment of cancer cells with the tRXRα-stabilizing compounds TRC5-7 resulted in enhanced AKT activity and apoptosis-resistance. However, although TR01 could strongly reduce tRXRα expression and AKT activity, it also strongly inhibited the expression and transcriptional activity of RXRα in normal cells. Importantly, the tRXRα-selective TRC4 that did not significantly inhibit RXRα transcriptional function retained the most potency of the anticancer effect of TR01 and had no significant effect on the viability of normal cells. In conclusion, our results demonstrated that tRXRα-selective TRC4 will have potential clinical application in terms of drug target and side effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.

A study on blocking store-operated Ca2+ entry in pulmonary arterial smooth muscle cells with xyloketals from marine fungi.

In this study, the effect of four xyloketals 1-4 on store-operated calcium entry (SOCE) was investigated in primary distal pulmonary arterial smooth muscle cells (PASMCs) isolated from mice. The results showed that xyloketal A (1), an unusual ketal with C-3 symmetry, exhibited strong SOCE blocking activity. Secretion of interleukin-8 (IL-8) was also inhibited by xyloketal A. The parallel artificial membrane permeability assay (PAMPA) of 1-4 suggested that these xyloketals penetrated easily through the cell membrane. Moreover, the molecular docking study of xyloketal A with activation region of the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1 (STIM1-ORAI1) protein complex, the key domain of SOCE, revealed that xyloketal A exhibited a noncovalent interaction with the key residue lysine 363 (LYS363) in the identified cytosolic regions in STIM1-C. These findings provided useful information about xyloketal A as a SOCE inhibitor for further evaluation.

Xyloketal B attenuates atherosclerotic plaque formation and endothelial dysfunction in apolipoprotein e deficient mice.

Our previous studies demonstrated that xyloketal B, a novel marine compound with a unique chemical structure, has strong antioxidant actions and can protect against endothelial injury in different cell types cultured in vitro and model organisms in vivo. The oxidative endothelial dysfunction and decrease in nitric oxide (NO) bioavailability are critical for the development of atherosclerotic lesion. We thus examined whether xyloketal B had an influence on the atherosclerotic plaque area in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet and investigated the underlying mechanisms. We found in our present study that the administration of xyloketal B dose-dependently decreased the atherosclerotic plaque area both in the aortic sinus and throughout the aorta in apoE-/- mice fed a high-fat diet. In addition, xyloketal B markedly reduced the levels of vascular oxidative stress, as well as improving the impaired endothelium integrity and NO-dependent aortic vasorelaxation in atherosclerotic mice. Moreover, xyloketal B significantly changed the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and Akt without altering the expression of total eNOS and Akt in cultured human umbilical vein endothelial cells (HUVECs). Here, it increased eNOS phosphorylation at the positive regulatory site of Ser-1177, while inhibiting phosphorylation at the negative regulatory site of Thr-495. Taken together, these findings indicate that xyloketal B has dramatic anti-atherosclerotic effects in vivo, which is partly due to its antioxidant features and/or improvement of endothelial function.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Secondary metabolites of a mangrove endophytic fungus Aspergillus terreus (No. GX7-3B) from the South China Sea.

The mangrove endophytic fungus Aspergillus terreus (No. GX7-3B) was cultivated in potato dextrose liquid medium, and one rare thiophene compound (1), together with anhydrojavanicin (2), 8-O-methylbostrycoidin (3), 8-O-methyljavanicin (4), botryosphaerone D (5), 6-ethyl-5-hydroxy-3,7-dimethoxynaphthoquinone (6), 3ß,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (7), 3ß,5α,14α-trihydroxy-(22E,24R)-ergosta-7, 22-dien-6-one (8), NGA0187 (9) and beauvericin (10), were isolated. Their structures were elucidated by analysis of spectroscopic data. This is the first report of a natural origin for compound 6. Moreover, compounds 3, 4, 5, 7, 8 and 10 were obtained from marine microorganism for the first time. In the bioactive assays in vitro, compounds 2, 3, 9 and 10 displayed remarkable inhibiting actions against α-acetylcholinesterase (AChE) with IC50 values 2.01, 6.71, 1.89, and 3.09 µM, respectively. Furthermore, in the cytotoxicity assays, compounds 7 and 10 exhibited strong or moderate cytotoxic activities against MCF-7, A549, Hela and KB cell lines with IC50 values 4.98 and 2.02 (MCF-7), 1.95 and 0.82 (A549), 0.68 and 1.14 (Hela), and 1.50 and 1.10 µM (KB), respectively; compound 8 had weak inhibitory activities against these tumor cell lines; compounds 1, 2, 3, 4, 5, 6 and 9 exhibited no inhibitory activities against them.

Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish.

We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.

Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and human endothelial cells via PI3K/Akt/eNOS signaling pathways.

Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.

Synthesis and DNA-binding affinities of protoberberine-based multivalent agents.

Three novel berberine derivatives bearing two, three, and four primary amino groups at C(9), respectively, were synthesized and characterized on the basis of ¹H- and ¹³C-NMR, MS, and HR-MS data. Their non-covalent binding with calf thymus (CT) DNA was investigated by means of spectrophotometric titration and ethidium bromide (EB) displacement experiments. The results indicated that these multivalent berberine derivatives exhibited up to 130-fold enhanced binding affinities relative to berberine, and thus, may be exploitable as potent DNA-binding agents.

Marine compound Xyloketal B protects PC12 cells against OGD-induced cell damage.

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Recently, we have demonstrated that Xyloketal B is an antioxidant and can protect against oxidized low density lipoprotein (LDL)-induced cell injury. In the present study, we investigated whether Xyloketal B can protect against ischemia-induced cell injury in an in vitro oxygen glucose deprivation (OGD) model of ischemic stroke in PC12 cells. We found that Xyloketal B could directly scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and protect PC12 cells against OGD insult. Furthermore, Xyloketal B alleviated OGD-induced mitochondria superoxide, mitochondria fragmentation and GTPase dynamin-related protein 1 (Drp1) overexpression as well as reduction of mitochondrial membrane potential. All together, the present study demonstrates that Xyloketal B protects PC12 cells against OGD-induced cell injury and that the anti-oxidative property and protective action on mitochondria may account for its neuroprotective actions.

A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro.

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Pretreatment with xyloketal B (0.63-40 microM) significantly improved oxLDL (150 microg/ml)-induced injury in human umbilical vein endothelial cells (HUVECs) without either toxic or proliferative effects. Xyloketal B concentration-dependently attenuated oxLDL-induced ROS generation, peroxynitrite formation and decrease of Bcl-2 expression. In addition, xyloketal B significantly inhibited NADPH oxidase activity, as well as mRNA expression of gp91phox and p47phox. Furthermore, xyloketal B alone augmented the production of nitric oxide (NO). Collectively, these data indicate that xyloketal B protects against oxLDL-induced endothelial oxidative injury probably through inhibiting NADPH oxidase-derived ROS generation, promoting NO production and restoring Bcl-2 expression, making it a promising compound for further evaluation in the treatment of atherosclerosis.

[Induction of G2 /M phase arrest and apoptosis of MCF-7 cells by novel benzofuran lignan via suppressing cell cycle proteins].

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids.

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.

DNA-binding affinities and sequence specificities of protoberberine alkaloids and their demethylated derivatives: a comparative study.

Berberrubine (1a), jatrorubine (2a), and palmatrubine (3a) have been chemically prepared by partial demethylation of berberine (1), jatrorrhizine (2), and palmatine (3), respectively. Their interactions with calf thymus (CT) DNA, poly(dA-dT)poly(dA-dT), poly(dG-dC)poly(dG-dC), and eight AT-rich 12-mer double-stranded DNAs have been investigated by means of competitive ethidium bromide (EB) displacement experiments. The results showed that DNA-binding affinities of these protoberberine alkaloids have been significantly improved by partial demethylation, and that all of these alkaloids have the preferable binding affinities with AT-rich DNA. Especially, the sequence specificities of DNA-binding of demethylated derivatives 1a, 2a, and 3a had changed to a certain extent when compared with the parent alkaloids 1, 2, and 3, respectively. The binding mode of these alkaloids was further confirmed by UV spectroscopic titration experiments. All the compounds bind to double-stranded DNA most probably via an intercalating mode.

Amplification of DNA-binding affinities of protoberberine alkaloids by appended polyamines.

This communication describes a synthetic approach toward the amplification of the moderate DNA-binding affinities of protoberberine alkaloids. Specifically, three protoberberine derivatives bearing two to six primary amino groups at the 3- and 9-positions of protoberberine were synthesized and characterized by NMR ((1)H and (13)C) and HRMS. Studies on their affinities toward calf thymus (CT) DNA by ethidium bromide (EB) displacement and spectrophotometric titration experiments indicate that these polyamino protoberberines show more than 10(3)-fold enhanced DNA-binding affinities relative to palmatine and thus are exploitable as strong DNA-binders.

Cytotoxic activity and cytostatic mechanism of novel 2-arylbenzo[b]furans.

The aims of this study were to screen cytotoxic compounds from 14 newly-synthesized 2-arylbenzo[b]furans and explore their mechanisms of action. Cytotoxicity was determined by the MTT method. Cell-cycle distribution was detected by flow cytometry. Wright-Giemsa staining was performed to demonstrate the morphological features of cells in mitotic phase. Polymerization of tubulin was detected by tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical study. Among the 14 compounds screened, 4-formyl-2-(4-hydroxy-3-methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo[b]furan (ERJT-12) showed significant cytotoxicity. Our results demonstrated that ERJT-12 exhibited anti-cancer activity in a variety of tumour cell lines with an IC50 value (concentration resulting in 50% inhibition of cell growth) of 5.75 approximately 17.29 microM. Cell cycle analysis showed a concentration-dependent accumulation of tumour cells in G2/M phase after treatment with ERJT-12. Further investigation indicated that ERJT-12 blocked the cell cycle in M phase, with separation and dispersion of chromosomes. ERJT-12 inhibited tubulin polymerization in-vitro. Changes of the cellular microtubule network caused by ERJT-12 were also detected, which were similar to the changes caused by colchicine. These results suggested that the anti-cancer activity of ERJT-12 is worth further investigation.