Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain.

Systemic toxicity and tumor cell resistance still limit the efficacy of chemotherapy in colorectal cancer. Therefore, alternative treatments are desperately needed. The thiazolide Nitazoxanide (NTZ) is an FDA-approved drug for the treatment of parasite-mediated infectious diarrhea with a favorable safety profile. Interestingly, NTZ and the thiazolide RM4819-its bromo-derivative lacking antibiotic activity-are also promising candidates for cancer treatment. Yet the exact anticancer mechanism(s) of these compounds still remains unclear. In this study, we systematically investigated RM4819 and NTZ in 2D and 3D colorectal cancer culture systems. Both compounds strongly inhibited proliferation of colon carcinoma cell lines by promoting G1 phase cell cycle arrest. Thiazolide-induced cell cycle arrest was independent of the p53/p21 axis, but was mediated by inhibition of protein translation via the mTOR/c-Myc/p27 pathway, likely caused by inhibition of mitochondrial respiration. While both thiazolides demonstrated mitochondrial uncoupling activity, only RM4819 inhibited the mitochondrial respiratory chain complex III. Interestingly, thiazolides also potently inhibited the growth of murine colonic tumoroids in a comparable manner with cisplatin, while in contrast to cisplatin thiazolides did not affect the growth of primary intestinal organoids. Thus, thiazolides appear to have a tumor-selective antiproliferative activity, which offers new perspectives in the treatment of colorectal cancer.

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis.

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.

Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts.

Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1.

Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.

Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein.

Selective degradation of proteins at the endoplasmic reticulum (ER-associated degradation) is thought to proceed largely via the cytosolic ubiquitin-proteasome pathway. Recent data have indicated that the dislocation of short-lived integral-membrane proteins to the cytosolic proteolytic system may require components of the Sec61 translocon. Here we show that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. In yeast mutants expressing functionally attenuated proteasomes, degradation of a short-lived doubly membrane-spanning protein proceeds rapidly through the N-terminal cytosolic domain of the substrate, but slows down considerably when continued degradation of the molecule requires membrane extraction. Thus, proteasomes engaged in ER degradation can directly process transmembrane proteins through a mechanism in which the dislocation of the substrate and its proteolysis are coupled. We therefore propose that the retrograde transport of short-lived substrates may be driven through the activity of the proteasome.