Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.     

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a–r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC₅₀ values ranging from 0.87 to 16.59 μM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC₅₀ values 0.91 and 0.87 μM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G₂/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G₂/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.     

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨ_m) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨ_m, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers

A series of 1,2,3-triazole linked aminocombretastatin conjugates were synthesized and evaluated for cytotoxicity, inhibition of tubulin polymerization and apoptosis inducing ability. Most of the conjugates exhibited significant anticancer activity against some representative human cancer cell lines and two of the conjugates 6d and 7c displayed potent cytotoxicity with IC₅₀ values of 53 nM and 44 nM against A549 human lung cancer respectively, and were comparable to combretastatin A-4 (CA-4). SAR studies revealed that 1-benzyl substituted triazole moiety with an amide linkage at 3-position of B-ring of the combretastatin subunit are more active compared to 2-position. G2/M cell cycle arrest was induced by these conjugates 6d and 7c and the tubulin polymerization assay (IC₅₀ of 1.16 μM and 0.95 μM for 6d and 7c, respectively) as well as immunofluorescence analysis showed that these conjugates effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Colchicine competitive binding assay suggested that these conjugates bind at the colchicine binding site of tubulin as also observed from the docking studies. Further, mitochondrial membrane potential, ROS generation, caspase-3 activation assay, Hoechst staining and DNA fragmentation analysis revealed that these conjugates induce cell death by apoptosis.     

Viridiflorol induces anti-neoplastic effects on breast,lung, and brain cancer cells through apoptosis

All active natural molecules are not fully exploited as therapeutic agents, causing delays in the advancement of anticancer drug discovery. Viridiflorol is a natural volatile element that may work as anti-cancer compound. We tested the anticancer properties of viridiflorol at different concentrations ranging from 0.03 to 300 μM in vitro on three cancer cells including breast (MCF-7), lung (A549) and brain (Daoy). The cancer cells responses were documented after treatment using MTT and Annexin V assays. Viridiflorol showed cytotoxic effects against all tested cell lines, reducing cell viability in a concentration-dependent manner with variable IC₅₀ values. Daoy and A549 cell lines were more sensitive to viridiflorol when compared with temozolomide and doxorubicin, respectively. Viridiflorol demonstrated the highest anticancer activity against the Daoy cells with an estimated IC₅₀ of 0.1 µM followed by MCF-7 at 10 µM, and A549 at 30 µM. In addition, upon exposure to concentrations ranging from 30 µM to 300 µM of viridiflorol, early and late apoptotic cell death was induced in a concentration dependent manner in Daoy (55.8%-72.1%), MCF-7 (36.2%-72.7%) and A459 (35%-98.9%) cell lines, respectively. In conclusion, viridiflorol demonstrates cytotoxic and apoptotic ability in three different cancer cell lines (brain, breast and lung).     

Synthesis and antiproliferative evaluation of 6-arylindeno[1,2-c]quinoline derivatives

A number of 6-arylindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antiproliferative activities against the growth of five cancer cell lines including human hepatocelluar carcinoma (Hep G2, Hep 3B and Hep2.2.1), non-small cell lung cancer (A549 and H1299), and normal diploid embryonic lung cell line (MRC-5). The preliminary results indicated that 9-(3-(dimethylamino)propoxy)-6-(4-(3-(dimethylamino)propoxy)phenyl)-2-fluoro-11H-indeno[1,2-c]quinolin-11-one (14c) was the most potent with GI₅₀ values of 0.61, 0.67, 0.59, and 0.72 μM against the growth of Hep G2, Hep 3B, Hep 2.2.1, and H1299 cells, respectively. Results have also shown that 2,9-bis(3-(dimethylamino)propoxy)-6-(4-(3-(dimethylamino)propoxy)phenyl)-11H-indeno[1,2-c]quinolin-11-one (17), which exhibited GI₅₀ of 0.60 and 0.68 μM against the growth of Hep G2 and A549, respectively, was more active than the positive topotecan and irinotecan. Compound 17 was less toxic than topotecan against the growth of normal cell (MRC-5) and therefore, was selected for further evaluation. Results indicated that compound 17 induce cell cycle arrest in G2/M phase, DNA fragmentation, and disrupt the microtubule network in A549 cells. The apoptotic induction may through the cleavage of PARP.     

Microtubules,microtubule-interfering agents and apoptosis

Microtubules are dynamic polymers that play crucial roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of anticancer drugs. Microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M phase, and late apoptosis. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will reveal sites of potential intervention in cancer chemotherapy. Inhibition of microtubule dynamics induces persistent modification of biological processes (M arrest) and signaling pathways (mitotic spindle assembly checkpoint activation, Bcl-2 phosphorylation, c-Jun NH₂-terminal kinase activation), which ultimately lead to apoptosis through the accumulation of signals that finally reach the threshold for the onset of apoptosis or through diminishing the threshold for engagement of cell death. Microtubules serve also as scaffolds for signaling molecules that regulate apoptosis, such as Bim and survivin, and their release from microtubules affect the activities of these apoptosis regulators. Thus, sustained modification of signaling routes and changes in the scaffolding properties of microtubules seem to constitute two major processes in the apoptotic response induced by microtubule-interfering agents.     

Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells

Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K. pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K. pneumoniae (Kp) gene ytfL as non‐pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K. pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia.     

Ionizing Radiation Potentiates Dihydroartemisinin-Induced Apoptosis of A549 Cells via a Caspase-8-Dependent Pathway

This report is designed to explore the molecular mechanism by which dihydroartemisinin (DHA) and ionizing radiation (IR) induce apoptosis in human lung adenocarcinoma A549 cells. DHA treatment induced a concentration- and time-dependent reactive oxygen species (ROS)-mediated cell death with typical apoptotic characteristics such as breakdown of mitochondrial membrane potential (Δψ_m), caspases activation, DNA fragmentation and phosphatidylserine (PS) externalization. Inhibition of caspase-8 or -9 significantly blocked DHA-induced decrease of cell viability and activation of caspase-3, suggesting the dominant roles of caspase-8 and -9 in DHA-induced apoptosis. Silencing of proapoptotic protein Bax but not Bak significantly inhibited DHA-induced apoptosis in which Bax but not Bak was activated. In contrast to DHA treatment, low-dose (2 or 4 Gy) IR induced a long-playing generation of ROS. Interestingly, IR treatment for 24 h induced G2/M cell cycle arrest that disappeared at 36 h after treatment. More importantly, IR synergistically potentiated DHA-induced generation of ROS, activation of caspase-8 and -3, irreparable G₂/M arrest and apoptosis, but did not enhance DHA-induced loss of Δψ_m and activation of caspase-9. Taken together, our results strongly demonstrate the remarkable synergistic efficacy of combination treatment with DHA and low-dose IR for A549 cells in which IR potentiates DHA-induced apoptosis largely by enhancing the caspase-8-mediated extrinsic pathway.     

Study of the redox properties of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and its glutathionyl conjugate in biological reactions: one- and two-electron enzymatic reduction

Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), the basic unit of several tetracyclic antitumor antibiotics, and its glutathione conjugate were reduced by the one- and two-electron transfer flavoproteins NADPH-cytochrome P450 reductase and DT-diaphorase to their semi- and hydroquinone forms, respectively. Kinetic studies performed on purified DT-diaphorase showed the following results: KNADPHm = 68 microM, KQuinonem = 0.92 microM, and Vmax 1300 nmol X min-1 X microgram enzyme-1. Similar studies performed on purified NADPH-cytochrome P450 reductase indicated a lower KNADPHm (10.5 microM) and higher KQuinonem (2.3 microM). The Vmax values were 20-fold lower (46 nmol X min-1 X micrograms enzyme-1) than those observed with DT-diaphorase. DT-diaphorase reduced the naphthazarin-glutathione conjugate with an efficiency 5-fold lower than that observed with the parent quinone. The nucleophilic addition of GSH to naphthazarin proceeded with GSH consumption at rates slower than those observed with 1,4-naphthoquinone and its monohydroxy derivative, 5-hydroxy-1,4-naphthoquinone. The initial rate of GSH consumption during these reactions did not vary whether the assay was carried out under anaerobic or aerobic conditions. Autoxidation accompanied the DT-diaphorase and NADPH-cytochrome P450 reductase catalysis of naphthazarin and its glutathionyl adduct as well as the 1,4-reductive addition of GSH to naphthazarin. Superoxide dismutase at catalytic concentrations (nM range) enhanced slightly (1.1- to 1.6-fold) the autoxidation following the enzymatic catalysis of naphthazarin. Autoxidation during the GSH reductive addition to 1,4-naphthoquinones decreased with increasing number of -OH substituents, 1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4-naphthoquinone, thus revealing that the contribution of redox transitions other than autoxidation, e.g., cross-oxidation, to the decay of the primary product of nucleophilic addition increases with increasing number of -OH substituents. Superoxide dismutase enhanced substantially the autoxidation of glutathionyl-naphthohydroquinone adducts, thereby affecting only slightly the total GSH consumed and GSSG formed during the reaction. The present results are discussed in terms of the relative contribution of one- and two-electron transfer flavoproteins to the bioreductive activation of naphthazarin and its glutathionyl conjugate as well as the importance of autoxidation reactions in the mechanism(s) of quinone cytotoxicity.     

New antiproliferative 7-(4-(N-substituted carbamoylmethyl)piperazin-1-yl) derivatives of ciprofloxacin induce cell cycle arrest at G2/M phase

New N-4-piperazinyl derivatives of ciprofloxacin 2a–g were prepared and tested for their cytotoxic activity. The primary in vitro one dose anticancer assay experienced promising cytotoxic activity against different cancer cell lines especially non-small cell lung cancer. Independently, compounds 2b, 2d, 2f and 2g showed anticancer activity against human non-small cell lung cancer A549 cells (IC₅₀ = 14.8, 24.8, 23.6 and 20.7 μM, respectively) compared to the parent ciprofloxacin (IC₅₀ >100 μM) and doxorubicin as a positive control (IC₅₀ = 1 μM). The flow cytometric analysis for 2b showed dose dependent G₂/M arrest in A549 cells. Also, 2b increased the expression of p53 and p21 and decreased the expression of cyclin B1 and Cdc2 proteins in A549 cells without any effect on the same proteins expression in WI-38 cells. Specific inhibition of p53 by pifithrin-α reversed the G₂/M phase arrest induced by the 2b compound, suggesting contribution of p53 to increase. Taken together, 2b induced G₂/M phase arrest via p53/p21 dependent pathway. The results indicate that 2b can be used as a lead compound for further development of new derivatives against non-small cell lung cancer.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72 h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC₅₀ values in the range of 0.31 (1.7 μM) in HL-60 to 0.88 μg/mL (4.7 μM) in SF-295 and IC₅₀ of 0.69 μg/mL (3.7 μM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC₅₀ significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.     

Induction of p53‐independent growth inhibition in lung carcinoma cell A549 by gypenosides

The objectives of this study are to investigate antiproliferative effect and mechanisms of bioactive compounds from Gynostemma pentaphyllum (G. pentaphyllum) on lung carcinoma cell A549. Saponins, carotenoids and chlorophylls were extracted and fractionated by column chromatography, and were subjected to high‐performance liquid chromatography‐mass spectrometry analyses. The saponin fraction, which consisted mainly of gypenoside (Gyp) XXII and XXIII, rather than the carotenoid and chlorophyll ones, was effective in inhibiting A549 cell growth in a concentration‐ and a time‐dependent manner as evaluated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The estimated half maximal inhibitory concentration (IC₅₀) of Gyp on A549 cells was 30.6 μg/ml. Gyp was further demonstrated to induce an apparent arrest of the A549 cell cycle at both the S phase and the G2/M phase, accompanied by a concentration‐ and a time‐dependent increase in the proportions of both the early and late apoptotic cells. Furthermore, Gyp down‐regulated cellular expression of cyclin A and B as well as BCL‐2, while up‐regulated the expression of BAX, DNA degradation factor 35 KD, poly [ADP‐ribose] polymerase 1, p53, p21 and caspase‐3. Nevertheless, both the treatment of a p53 inhibitor, pifithrin‐α, and the small hairpin RNA‐mediated p53 knockdown in the A549 cells did not alter the growth inhibition effect induced by Gyp. As a result, the cell cycle arrest and apoptosis of A549 cells induced by Gyp would most likely proceed through p53‐independent pathway(s).     

Novel hybrids of podophyllotoxin and formononetin inhibit the growth,migration and invasion of lung cancer cells

In this study, three hybrids of podophyllotoxin and formononetin were synthesized and evaluated for anticancer efficacy. Some of the derivatives exhibited potent cytotoxicity against a panel of human and mouse cancer cell lines, with IC₅₀ values in the low micromolar to submicromolar range. Evaluation against A549 lung tumor cell line identified that the IC₅₀ value of compound 10a was 0.753 μM, indicating that 10a was 2.568-fold more efficacious than parent podophyllotoxin. Mechanistic studies revealed that 10a induced A549 cell apoptosis mainly via caspase pathway, as well as disrupted the microtubule organization by occupying the colchicine binding site of the tubulin. Moreover, wound healing assay and transwell invasion assay indicated that 10a displayed potent inhibitory effects on invasion and migration in A549 cancer cells. In additiona, a decrease in vimentin immunostaining was also observed in A549 cells after treatment with 10a. Overall, hybrid 10a might be a promising candidate for the potential treatment of human lung carcinoma.     

Organization of cortical microtubules at the plasma membrane in Arabidopsis

 To understand the role of microtubules in the regulation of cell elongation, we characterized microtubule patterns in fass, a cell shape mutant of Arabidopsis thaliana (L.) Heynh. Examining microtubule patterns via immunocytochemistry, we found that fass cells were able to organize their microtubules into mitotic spindles and phragmoplasts. During interphase or preprophase, fass cells had cortical microtubules, verified by transmission electron microscopy, but these microtubules were not organized into the cortical array or preprophase band. Using chromatin condensation and tubulin localization on the nuclear envelope as preprophase stage markers, we found that although fass cells lacked the preprophase band and cortical array, their cell division cycle appeared normal. To pinpoint the defect in fass cells, we delineated the sequential events leading to cortical array formation in Arabidopsis cells and found that fass cells initiated and recolonized cortical microtubules in the same manner as wild-type cells, but failed to order them into the cortical array. Taken together, these results suggest fass cells are impaired in a component of the microtubule organizing center(s) required for the proper ordering of cortical microtubules at the plasma membrane. Received: 23 August 1996 / Accepted: 25 September 1996