The sponge-derived polyketide macrolides fijianolides A (1) and B (2), isolaulimalide and laulimalide, have taxol-like microtubule-stabilizing activity, and the latter exhibits potent cytotoxicity. Insight on the biogeographical and phenotypic variations of Cacospongia mycofijiensis is presented that will enable a future study of the biosynthetic pathway that produces the fijianolides. In addition to fijianolides A and B, six new fijianolides, D-I (7-12), were isolated, each with modifications to the C-20 side chain of the macrolide ring. Compounds 7-12 exhibited a range of in vitro activities against HCT-116 and MDA-MB-435 cell lines. Fijianolides 8 and 10 were shown to disrupt interphase and mitotic division, but were less potent than 2. An in vivo evaluation of 2 using tumor-bearing severe combined immuno-deficiency mice demonstrated significant inhibition of growth in HCT-116 tumors over 28 days.

A multidimensional chromatographic 2-D liquid-phase separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of protein expression between Peninsularinone-treated and untreated HCT-116 human colon adenocarcinoma cells. The method involves fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous reversed-phase HPLC. A 2-D map of the protein content of each cell line based upon pI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or downregulated proteins displayed using ProteoVue and DeltaVue software. Using this method, > 1000 protein bands could be detected in 0.2 pH fractions over a pH range of 4-7. In addition, the liquid eluent from the separation was directed on-line into an electrospray TOF-MS to obtain an accurate molecular weight of the intact proteins. An accurate molecular weight together with the peptide map was used to obtain protein identification using database searching. The method has been shown to have high reproducibility for quantitative differential display analysis of interlysate comparisons, generation of accurate protein identifications, and ease of data interpretation. It has been used herein to identify proteins that change as a function of drug treatment. The relative simplicity of the current procedure and the potential for full automation will make this technique an essential tool in future proteomic studies.

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XK469 (NSC 697887) is a novel antitumor agent with broad activity against a variety of tumors. Previous studies suggest that XK469 is a topoisomerase II␤ poison with functional activity similar to that of 4-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The goal of our study was to investigate its mechanism of action further using a human HCT-116 (H116) colon tumor cell model. Concentrationsurvival curves with continuous exposure indicated that XK469 had low cytotoxic activity against H116 cells. Cell cycle analysis revealed that XK469 is a phase-specific cell cycle blocker that is associated with increased levels of cyclin B1, cyclin A and p53 but not CDK1 (cdc2) or cyclin E. In contrast, treatment of H116 cells with m-AMSA caused a total degradation of both cyclin A and B1 but enhanced expression of cyclin E and p53. Accumulation of cyclin B1 in XK469-treated cells was correlated with the inhibition of cyclin B1 ubiquitination, a metabolic process mandatory for proteasome-mediated protein turnover. However, no inhibition of cyclin B1 ubiquitination was detected in cells treated with m-AMSA or colchicine, a known mitotic inhibitor. Furthermore, unlike m-AMSA, XK469 did not induce caspase activation or apoptotic cell death in H116 cells. Our results suggest that XK469 is a phase-specific cell cycle inhibitor with a unique mechanism of action that is correlated with the inhibition of cyclin B1 ubiquitination and its accumulation at early M phase. © 2002 Wiley-Liss, Inc. Key words: antitumor; cyclin B1; ubiquitination; XK469XK469 (NSC 697887), a synthetic quinoxaline phenoxypropionic acid derivative, has been found to have broad range of activity against a variety of tumors, including multiple drug-resistant (MDR)-expressing solid tumors. Initially, XK469 was selected through a disk-diffusion soft agar colony-formation assay and was found to have selective antiproliferative activity for solid tumors. 1,2 Despite the demonstration of impressive activity in vivo, very little is known about its molecular targets and mechanism of action. A recent study by Gao et al. 3 showed that XK469 might be a selective topoisomerase II␤ poison with functional activity similar to that of the topoisomerase II inhibitor 4Ј-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). Like m-AMSA, both XK469 and its S-and R-isomers induce reversible protein-DNA crosslinks in mammalian cells. 3 However, recent studies using H116 human colon carcinoma cells in our laboratories suggest that XK469 is a phase-specific cell cycle inhibitor acting at early M phase with low antiproliferative effect. 4 This finding raises the possibility that cell cycle regulators are involved in mediating the effect of XK469.Molecules involved in the basic machinery of the cell cycle, which include cyclins, cyclin-dependent kinases (CDKs), kinase inhibitors and related phosphatases that regulate CDKs, have been isolated and characterized. 5,6 For orderly cell division, CDKs have to be activated and inactivated in an intricate manner at specific time points ...

Our findings suggest that the antiproliferative effect of XK469 is mediated by inhibiting the MEK/MAPK signaling pathways in U-937 human leukemia cells.

The cellular paradigm presented here defines the cellular action profile of new anticancer agents that complements our discovery and development paradigm. The main elements of this profile include a concentration clonogenicity response relationship on proliferating and plateau phase cells, flow cytometry studies assessing progression delay and apoptosis, macromolecular synthesis inhibition, and DNA damage assessment by the comet assay; other specific assessments then derive from these findings such as topoisomerase assays. XK469 is a new anticancer agent derived from the herbicide Assure that is the inactive parent compound of a family of quinoxaline analogs found to have anticancer activity in vivo. We have applied the described cellular action profile paradigm to XK469 to define a novel action at the cellular level. XK469 is a G2M phase-specific, antiproliferative agent whose activity is related to the 7-position of the chlorine ion in the benzene ring and expressed through a unique cellular action profile resulting in the irreversible increase in cyclin B1 (possibly by specific inhibition of its ubiquitination) and leading, in the absence of apoptosis, to the final mitotic arrest of HCT-116 cells in prophase with subsequent loss of clonogenicity.

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