Microtubule-interfering Agents Research Articles

EM012, a microtubule-interfering agent, inhibits the progression of multidrug-resistant human ovarian cancer both in cultured cells and in athymic nude mice

Drug resistance, in particular multidrug resistance, is a serious problem that impedes the effectiveness of chemotherapy. Multidrug resistance results mainly from an enhanced efflux of drugs by drug pumps located on the cell membrane such as P-glycoprotein. In the study reported here we showed that EM012, a microtubule-interfering agent, is a weak substrate for P-glycoprotein and inhibited the proliferation of A2780/ADR human ovarian cancer cells, which possess multidrug resistance due to P-glycoprotein overexpression. A2780/ADR cells treated with EM012 exhibited pronounced mitotic arrest, developed large multilobed nuclei, and eventually died through the initiation of apoptosis. Intraperitoneal treatment of A2780/ADR xenograft tumors in athymic nude mice with EM012 significantly inhibited tumor progression through triggering apoptosis and conferred an apparent survival advantage. Furthermore, EM012 treatment did not cause detectable toxicity to normal tissues. These findings suggest that EM012 may serve as a novel chemotherapeutic agent for the treatment of multidrug-resistant human ovarian cancer.

Pharmacological and small interference RNA‐mediated inhibition of breast cancer‐associated fatty acid synthase (oncogenic antigen‐519) synergistically enhances Taxol (paclitaxel)‐induced cytotoxicity

The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.

ω-6 polyunsaturated fatty acid γ-linolenic acid (18:3n-6) enhances docetaxel (Taxotere) cytotoxicity in human breast carcinoma cells: Relationship to lipid peroxidation and HER-2/neu expression

The omega-6 polyunsaturated fatty acid gamma-linolenic acid (GLA; 18:3n-6) has raised recent interest as novel anti-cancer agent as it possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. The taxane docetaxel (Taxotere) is currently one of the most active microtubule-interfering agents for breast cancer. Despite this encouraging therapeutical potential, the clinical use of taxanes involves problems related to the solubility, toxicity and development of drug resistance, which may be partially dependent on the expression of HER-2/neu oncogene. Current trends in the treatment of human tumors are for drug combinations that result in improved responses as well as the ability to use less toxic concentrations of the drugs. Here, we examined the cytotoxic effects of GLA in combination with docetaxel against estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231 and SK-Br3) human breast carcinoma cell lines. The cells were exposed simultaneously to GLA and docetaxel or sequentially to GLA followed by docetaxel for 24 h. Cytotoxicity was evaluated by the MTT assay, and the nature of the interactions between GLA and docetaxel (antagonism, additivity, and synergism) was analyzed by median effect and isobologram analyses. Interaction assessment showed that concurrent exposure to GLA plus docetaxel for 24 h resulted in synergism for MCF-7 and MDA-MB-231 cells, whereas an additive effect was observed in SK-Br3 cells. When exposure to GLA (24 and 48 h) was followed sequentially by docetaxel (24 h) a synergistic effect was observed in MDA-MB-231 and SK-Br3 cells, whereas an additive effect was found in MCF-7 cells. GLA-mediated increase in docetaxel cytotoxicity was only marginally abolished by Vitamin E, a lipid peroxidation inhibitor. Moreover, simultaneous exposure to GLA and docetaxel in the presence of the anti-oxidant Vitamin E also resulted in synergism, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of docetaxel-induced cytotoxicity. Further experiments showed that GLA markedly decreased the expression of p185HER-2/neu oncoprotein in MCF-7 breast cancer cells (</=85%), and RT-PCR analysis revealed that HER-2/neu mRNA was selectively decreased in a concentration-dependent manner following GLA treatment. Therefore, our results show that the fatty acid GLA enhances the cytotoxicity of docetaxel in human breast cancer cells by mechanisms other than lipoperoxidation, and that GLA-induced transcriptional repression of HER-2/neu oncogene might be one component of the mechanisms of this interaction.

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN).

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

Brominated derivatives of noscapine are potent microtubule-interfering agents that perturb mitosis and inhibit cell proliferation.

Noscapine, a microtubule-interfering agent, has been shown to arrest mitosis, to induce apoptosis, and to have potent antitumor activity. We report herein that two brominated derivatives of noscapine, 5-bromonoscapine (5-Br-nosc) and reduced 5-bromonoscapine (Rd 5-Br-nosc), have higher tubulin binding activity than noscapine and affect tubulin polymerization differently from noscapine. In addition, they are able to arrest cell cycle progression at mitosis at concentrations much lower than noscapine. Interestingly, whereas noscapine-arrested cells have nearly normal bipolar spindles, cells arrested by 5-Br-nosc and Rd 5-Br-nosc form multipolar spindles. Nevertheless, noscapine and the two derivatives all affect the attachment of chromosomes to spindle microtubules and they impair the tension across paired kinetochores to similar degrees. 5-Br-nosc and Rd 5-Br-nosc are also more active than noscapine in inhibiting the proliferation of various human cancer cells, including those that are resistant to paclitaxel and epothilone. Our results thus indicate a great potential for the use of 5-Br-nosc and Rd 5-Br-nosc both as biological tools for studying microtubule-mediated processes and as chemotherapeutic agents for the treatment of human cancers.

Nocodazole-induced p53-dependent c-Jun N-terminal Kinase Activation Reduces Apoptosis in Human Colon Carcinoma HCT116 Cells

Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53-/- cells in response to short term nocodazole treatment through application of Kinetworks immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53-/- cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53-/- cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53-/- cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function.

Gene expression profiling of exposure to TZT-1027, a novel microtubule-interfering agent, in non-small cell lung cancer PC-14 cells and astrocytes.

Clinical use of TZT-1027, a microtubule-interfering agent that inhibits the polymerization of tubulin, is expected because of its potent effects on solid tumors. TZT-1027 is thought to act directly on cellular microtubules, and arrest cell mitosis, however, the molecular mechanisms of the microtubule damage by TZT-1027 have not been fully identified. To investigate the possible novel mechanisms of action of TZT-1027, we used the cDNA macroarray technique to examine its effect on the expression of hundreds of tightly transcriptionally controlled genes. We used two cell lines, one was human non-small cell lung carcinoma PC-14 cells as a model for cancer cells, and the other was human astrocytes as a model for normal neuronal cells, because the dose-limiting-factor of microtubule-interfering agents is mainly peripheral neurotoxicity. mRNAs prepared from the PC-14 and astrocyte cell lines treated with TZT-1027 were compared with 588 genes spotted onto the filter, and which gene groups TZT-1027 modulated between the two cell lines was investigated. TZT-1027 exposure modulated expression of a variety of genes including the genes encoding cell-cycle and growth regulators, receptors, angiogenesis and invasion regulators, rho family small GTPases and their regulators and growth factors and cytokines. However, the way of gene regulation by TZT-1027 exposure was different between PC-14 cells and astrocytes. Genes up-regulated in both PC-14 cells and astrocytes were those for RAR-epsilon, TNFR 1 and 2 and so on. Specifically altered genes in PC-14 cells, such as the genes coding for cytokeratin 8, XPG, fau and the genes regulated only in PC-14 cells may be involved in the antitumor activity of TZT-1027. On the other hand, growth factor receptor precursors was upregulated specifically in astrocytes by TZT-1027 and this gene regulation only in astrocytes may be candidates related with neurotoxicity.

Inhibition of BRCA1 leads to increased chemoresistance to microtubule-interfering agents, an effect that involves the JNK pathway.

We have developed ribozymes (Rz) that inhibit BRCA1 expression in order to study the role of this gene in chemosensitivity. Two Rz, targeting positions 358 or 5282 of the BRCA1 mRNA, were cloned into the retroviral vector LXSN and lipofected into the breast cancer cell-line HBL100. We obtained 79-99% inhibition of BRCA1 expression, as determined by real-time quantitative PCR and by Western blotting. Decreased expression of BRCA1 led to sensitivity to the DNA damaging agents cisplatin and etoposide, resistance to the microtubule-interfering agents (MIA) taxol and vincristine. The molecular mechanism of resistance to MIA was investigated further by determining the status of the JNK pathway. We found that JNK1 expression was elevated, while JNK2 expression was decreased in Rz-expressing clones compared to controls. We have quantified the mRNA levels of BRCA1, JNK1, 2, MEK-4, -7 and c-jun after treatment with MIA. Vincristine treatment of control cells resulted in transcriptional repression of BRCA1, while the JNK1, 2, MEK-4, -7 and c-jun genes were induced. In Rz-treated cells, only JNK1 and MEK-4 were expressed and none was induced after MIA treatment. We then studied the phosphorylation of c-jun, a downstream effector of the JNK pathway. We observed a strong increase in phosphorylated c-jun after MIA treatment of the control cells but not in BRCA1-Rz treated cells, suggesting inhibition of the JNK pathway. These results show that the BRCA1-JNK pathway is involved in the cytotoxic response to MIA treatment, and inhibition of BRCA1 leads to transcriptional modifications of the JNK pathway.

Synergistic interaction between topotecan and microtubule-interfering agents.

Topotecan is a topoisomerase I inhibitor with demonstrated anticancer activity in preclinical and clinical studies. The purpose of the present study was to evaluate drug-drug interactions in therapeutic regimens that would combine topotecan with microtubule-interfering agents, such as Taxol and vinblastine. The cytotoxic activities of various drug combinations and schedules of administration were measured in a colon cancer cell line using the MTT assay. Western blot and flow cytometry were performed to determine the effects of Taxol and vinblastine on topoisomerase I and Bcl-xL protein levels and cell cycle distribution. Brief incubation of colon cancer cells with low concentrations of either Taxol or vinblastine increased the efficacy of a subsequent treatment with topotecan. Preincubation of cells with vinblastine or Taxol reduced by 10- to 40-fold the concentration of topotecan necessary to induce a 50% decrease in cell survival. The effects were maximal when the cells were treated for 5 h with microtubule-interfering agents and then incubated for 19 h in drug-free medium before the addition of topotecan. Under these conditions, both Taxol and vinblastine caused an increase in topoisomerase I protein levels, fraction of S phase cells, and extent of Bcl-xL phosphorylation immediately prior to the addition of topotecan. All these factors may contribute to the increased efficacy of topotecan observed with sequential therapy. Combinations of topotecan and microtubule-interfering agents result in synergistic anticancer activity when the drugs are administered sequentially. The promising preclinical data presented here encourage clinical testing of these drug combinations using a sequential schedule of administration.

Characterization of NR13-related human cell death regulator, Boo/ Diva, in normal and cancer tissues

Mouse Boo/ Diva is an ovary-specific member of the Bcl-2 family identified through homology with the avian cell death antagonist NR13. We identified a human orthologue of Boo/Diva, which is highly conserved between mouse and human and related to avian NR13. Human Boo/Diva is also expressed in human liver and kidney in addition to the ovary. We found that green fluorescence protein (EGFP)-tagged Boo/Diva was not exclusively localized to mitochondria before the induction of apoptosis. However, EGFP-Boo/Diva translocated to mitochondria in the process of apoptosis induced by vincristine, a microtubule-interfering agent. Overexpression of human Boo/Diva promoted cell death in HeLa and 293 cells. The cell death antagonist Bcl-XL interacts with Boo, but is unable to protect 293 cells from Boo/Diva-induced cell death. Finally, we mapped human Boo/ Diva to chromosome 15q21, a locus known to be related to human cervical cancer. Moreover, we found that genomic DNAs of three of 24 human cervical cancer samples display deletions within their Boo/ Diva genes. This result suggests a role for human Boo/ Diva in the pathogenesis of cervical cancer.

ChemInform Abstract: COBRA-1, a Rationally-Designed Epoxy-THF Containing Compound with Potent Tubulin Depolymerizing Activity as a Novel Anticancer Agent.

A novel mono-THF containing synthetic anticancer drug, COBRA-1, was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of α-tubulin. COBRA-1 inhibited GTP-induced tubulin polymerization in cell-free tubulin turbidity assays. Treatment of human breast cancer and brain tumor (glioblastoma) cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Like other microtubule-interfering agents, COBRA-1 activated the pro-apoptotic c-Jun N-terminal kinase (JNK) signal transduction pathway, as evidenced by rapid induction of c-jun expression.

COBRA-1, A rationally-designed epoxy-THF containing compound with potent tubulin depolymerizing activity as a novel anticancer agent

A novel mono-THF containing synthetic anticancer drug, COBRA-1, was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of α-tubulin. COBRA-1 inhibited GTP-induced tubulin polymerization in cell-free tubulin turbidity assays. Treatment of human breast cancer and brain tumor (glioblastoma) cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Like other microtubule-interfering agents, COBRA-1 activated the pro-apoptotic c-Jun N-terminal kinase (JNK) signal transduction pathway, as evidenced by rapid induction of c-jun expression.

Microtubule-interfering Agents Stimulate the Transcription of Cyclooxygenase-2: EVIDENCE FOR INVOLVEMENT OF ERK1/2 AND p38 MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS

We investigated whether microtubule-interfering agents (MIAs: taxol, colchicine, nocodazole, vinblastine, vincristine, 17-beta-estradiol, 2-methoxyestradiol) altered cyclooxygenase-2 (COX-2) expression in human mammary epithelial cells. MIAs enhanced prostaglandin E(2) synthesis and increased levels of COX-2 protein and mRNA. Nuclear run-off assays revealed increased rates of COX-2 transcription after treatment with MIAs. Calphostin C, an inhibitor of protein kinase C, blocked the induction of COX-2 by MIAs. The stimulation of COX-2 promoter activity by MIAs was inhibited by overexpressing dominant negative forms of Rho and Raf-1. MIAs stimulated ERK, JNK, and p38 mitogen-activated protein kinases (MAPK); pharmacological inhibitors of MAPK kinase and p38 MAPK blocked the induction of COX-2 by MIAs. Overexpressing dominant negative forms of ERK1 or p38 MAPK inhibited MIA-mediated activation of the COX-2 promoter. MIAs stimulated the binding of the activator protein-1 transcription factor complex to the cyclic AMP response element in the COX-2 promoter. A dominant negative form of c-Jun inhibited the activation of the COX-2 promoter by MIAs. Additionally, cytochalasin D, an agent that inhibits actin polymerization, stimulated COX-2 transcription by the same signaling pathway as MIAs. Thus, microtubule- or actin-interfering agents stimulated MAPK signaling and activator protein-1 activity. This led, in turn, to induction of COX-2 gene expression via the cyclic AMP response element site in the COX-2 promoter.

Ribozymes targeting the tumor suppressor BRCA1 lead to increased chemosensitivity to cisplatin and etoposide and chemoresistance to microtubule-interfering agents in HBL100 breast cancer cell line

Ribozymes targeting the tumor suppressor BRCA1 lead to increased chemosensitivity to cisplatin and etoposide and chemoresistance to microtubule-interfering agents in HBL100 breast cancer cell line

Activation of MAD 2 checkprotein and persistence of cyclin B1/CDC 2 activity associate with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells.

Paclitaxel (Taxol) is a microtubule-interfering agent that induced persistent and transient G2/M arrest before apoptosis in human nasopharyngeal carcinoma (NPC) cells at high and low concentrations, respectively. In this study, we intended to explore the underlying molecular events and found that cellular cyclin B1/CDC 2 kinase activity was increased and persisted for >6 h upon paclitaxel treatment both at high and low concentrations. Furthermore, activation of MAD 2 checkprotein could account for the loss of cyclin B1 ubiquitination and the persistence of cyclin B1/CDC 2 activation in the cases. To investigate the involvement of cyclin B1 and MAD 2 activation in paclitaxel-induced apoptosis, we introduced affinity-purified anti-cyclin B1 and MAD 2 antibodies into NPC cells by electroporation before the further paclitaxel treatment. The antibodies against cyclin B1 and MAD 2 indeed attenuated paclitaxel-induced cytotoxicity and DNA fragmentation. Our study suggests that activation of cyclin B1/CDC 2 and MAD 2 were the M-phase events required for paclitaxel-induced apoptosis in NPC cells. The dys-regulated cyclin B1/CDC 2 activation could enhance the prometaphase progression, but activation of MAD 2 rendered cells inable to exit from the metaphase. Under this circumstance, cells were probably going to "mitotic catastrophe" and ultimately, destined to apoptosis.

Induced differentiation in HT29, a human colon adenocarcinoma cell line.

The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.

Microtubule-interfering Agents Activate c-Jun N-terminal Kinase/Stress-activated Protein Kinase through Both Ras and Apoptosis Signal-regulating Kinase Pathways

The essential cellular functions associated with microtubules have led to a wide use of microtubule-interfering agents in cancer chemotherapy with promising results. Although the most well studied action of microtubule-interfering agents is an arrest of cells at the G2/M phase of the cell cycle, other effects may also exist. We have observed that paclitaxel (Taxol), docetaxel (Taxotere), vinblastine, vincristine, nocodazole, and colchicine activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway in a variety of human cells. Activation of JNK/SAPK by microtubule-interfering agents is dose-dependent and time-dependent and requires interactions with microtubules. Functional activation of the JNKK/SEK1-JNK/SAPK-c-Jun cascade (where JNKK/SEK1 is JNK kinase/SAPK kinase) was demonstrated by activation of a 12-O-tetradecanoylphorbol-13-acetate response element (TRE) reporter construct in a c-Jun dependent fashion. Microtubule-interfering agents also activated both Ras and apoptosis signal-regulating kinase (ASK1) and coexpression of dominant negative Ras and dominant negative apoptosis signal-regulating kinase exerted individual and additive inhibition of JNK/SAPK activation by microtubule-interfering agents. These findings suggest that multiple signal transduction pathways are involved with cellular detection of microtubular disarray and subsequent activation of JNK/SAPK.