A major drawback of cancer chemotherapy is the lack of tumor-specific targets which would allow for the selective eradication of malignant cells without affecting healthy tissues. In contrast with normal cells, most tumor cells contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. Many tumor cells regain mitotic stability after clonal selection by the coalescence of multiple centrosomes into two functional spindle poles. To overcome the limitations of current cancer treatments, we have developed a cell-based screening strategy to identify small molecules that inhibit centrosomal clustering and thus force tumor cells with supernumerary centrosomes to undergo multipolar mitoses, and subsequently, apoptosis. Using a chemotaxonomic selection of fungi from a large culture collection, a relatively small but diverse natural product extract library was generated. Screening of this compound library led to the identification of griseofulvin, which induced multipolar spindles by inhibition of centrosome coalescence, mitotic arrest, and subsequent cell death in tumor cell lines but not in diploid fibroblasts and keratinocytes with a normal centrosome content. The inhibition of centrosome clustering by griseofulvin was not restricted to mitotic cells but did occur during interphase as well. Whereas the formation of multipolar spindles was dyneinindependent, depolymerization of interphase microtubules seemed to be mechanistically involved in centrosomal declustering. In summary, by taking advantage of the tumorspecific phenotype of centrosomal clustering, we have developed a screening strategy that might lead to the identification of drugs which selectively target tumor cells and spare healthy tissues. [Cancer Res 2007;67(13):6342-50]
Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34 griseofulvin analogues as inhibitors of centrosomal clustering. The variations in the griseofulvin structure cover five positions, namely the 4, 5, 2', 3', and 4' positions. Modification of the 4 and 5 positions affords inactive molecules. The enol ether must be at the 2' position, and the 4' position needs to be sp(2) hybridized. The most active analogues were the 2'-benzyloxy and 2'-(4-methylbenzyloxy) analogues as well as the oxime of the former with a 25-fold increase of activity compared to griseofulvin. Comparison of the results obtained in this work with prior reported growth inhibition data for dermatophytic fungi showed both similarities and differences.
Vinblastine and vincristine are dimeric indole alkaloids derived from Catharanthus roseus (formerly: Vinca rosea). Their monomeric precursor molecules are vindoline and catharanthine. While vinblastine and vincristine are well-known mitotic spindle poisons, not much is known about vindoline and catharanthine. Vindoline and catharanthine showed weak cytotoxicity, while vinblastine, vincristine, and the semisynthetic vindesine and vinorelbine revealed high cytotoxicity towards cancer cells. This may reflect a general biological principle of poisonous plants. Highly toxic compounds are not only active towards predators, but also towards plant tissues. Hence, plants need mechanisms to protect themselves from their own poisons. One evolutionary strategy to solve this problem is to generate less toxic precursors, which are dimerized to toxic end products when needed. As shown by in silico molecular docking and biochemical approaches, vinblastine, vincristine and vinorelbine bound with high affinity to α/β-tubulin and inhibited tubulin polymerization, whereas the effects of vindoline and catharanthine were weak. Similarly, vinblastine produced high fractions of mono- and multipolar mitotic spindles, while vindoline and catharanthine did only weakly affect bipolar mitotic spindle formation. Here, we show that vinblastine contributes to cell death by interference with spindle polarity. P-glycoprotein-overexpressing multidrug-resistant CEM/VCR1000 cells were highly resistant towards vincristine and cross-resistant to vinblastine, vindesine, and vinorelbine, but not or only weakly cross-resistant to vindoline and catharanthine. In addition to tubulin as primary target, microarray-based mRNA signatures of responsiveness of these compounds have been identified by COMPARE and signaling pathway profiling.
BackgroundDasatinib is a multitargeted inhibitor of ABL, the SRC family, and other tyrosine kinases. We sought to evaluate the effects of this drug on cell proliferation, centrosomes, mitotic spindles, and cell cycle progression in vitro and in vivo.
The lack of tumor-specific targets that allow for selective eradication of malignant cells without affecting healthy tissues is a major drawback of cancer chemotherapy. In contrast to normal cells, tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. In many tumor types, mitotic stability is regained after clonal selection by coalescence of multiple centrosomes into two functional spindle poles (centrosomal clustering). To identify potent and selective inhibitors of centrosomal clustering, we recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res2007; 67: 6342–6350). We here describe the novel small molecule GF-15, which is a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas and multiple myeloma) as well as solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1–5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized tumor cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, short term treatment with GF-15 was also associated with inhibition of cytokine-triggered cell migration. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors.
300 The lack of tumor-specific targets that would allow for the selective eradication of malignant cells without affecting healthy tissues is a major challenge in the development of novel therapies for multiple myeloma (MM). In contrast to normal cells, malignant plasma cells frequently contain multiple centrosomes, associated with the transient formation of multipolar mitotic spindles that lead to segregation defects and chromosomal instability. As in most tumor types, mitotic stability in these cells is maintained by coalescence of multiple centrosomes into two functional spindle poles, termed “centrosomal clustering”. As we have recently shown, this mechanism is an attractive therapeutic target with specificity for tumor cells. To identify potent and selective inhibitors of centrosomal clustering, we performed a phenotype-based small molecule screen in order to force tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. We here describe the characterization of a novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis in MM cells. We tested a wide array of MM cell lines, including those resistant to conventional chemotherapeutic agents, and primary patient cells. We found mean inhibitory concentrations (IC50) of proliferation and survival in the range of 1-5 mM, associated with annexin V conversion and activation of caspases 8, 9, and 3. Importantly, GF-15 also overcomes the tumor cell growth advantage conferred by both bone marrow stromal cell-MM, and endothelial cell-MM, co-culture systems. Moreover, non-malignant cells without supernumerary centrosomes like activated PBMCs, immortalized hepatocytes, and bone marrow stromal cells did not reach their IC50 at doses of up to 50 mM. To further demonstrate the specificity of GF-15, we generated resistant MM cell lines by long-term culture with sub-IC50 doses of GF-15. In resistant cell lines, therapeutic doses of GF-15 no longer induce multipolar spindles, consistent with a significant loss of centrosomal aberrations in these cells, as observed by immunoflourescence microscopy. Mechanistically, cell cycle analysis of synchronized MM cells showed marked G2/M arrest within 12-16h followed by a dramatic increase of the sub-G1 fraction after treatment with GF-15. In addition, short term treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered MM cell migration. Co-treatment assays to assess potential partners for therapeutic combinations revealed at least additive effects for GF-15 together with bortezomib and marked synergism with paclitaxel at very low doses (1-5 nM), while the combination with melphalan resulted in antagonistic effects due to the S-phase arrest of tumor cells induced by melphalan. Finally and most importantly, i.p. as well as oral treatment of murine xenograft models of human MM resulted in tumor growth inhibition and significantly prolonged survival in vivo. Growth inhibition of xenograft tumor samples was associated with a dramatic increase of mitotic aberrations and multipolarity, as assessed by immunohistochemistry. In vivo biodistribution studies are ongoing. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a prototype small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and therefore strongly support its further evaluation and development as a lead compound of a new class of therapeutics for human malignancies. Disclosures: No relevant conflicts of interest to declare.
Supplementary Figure 3 from Identification of Griseofulvin as an Inhibitor of Centrosomal Clustering in a Phenotype-Based Screen
Supplementary Figure 1 from Identification of Griseofulvin as an Inhibitor of Centrosomal Clustering in a Phenotype-Based Screen
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.