Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex.

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating (a) cyclic AMP generation (Galphas), (b) calcium release (Galphaq), and (c) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Galphao/i and Galphaq). BIM-46174 inhibits the growth of a large panel of human cancer cell lines, including anticancer drug-resistant cells. Exposure of cancer cells to BIM-46174 leads to caspase-3-dependent apoptosis and poly(ADP-ribose) polymerase cleavage. National Cancer Institute COMPARE analysis for BIM-46174 supports its novel pharmacologic profile compared with 12,000 anticancer agents. The growth rate of human tumor xenografts in athymic mice is significantly reduced after administration of BIM-46174 combined with either cisplatin, farnesyltransferase inhibitor, or topoisomerase inhibitors. Our data validate the feasibility of targeting heterotrimeric G-protein functions downstream the GPCRs to improve anticancer chemotherapy.

Cytotoxicity of RH1: NAD(P)H:quinone acceptor oxidoreductase (NQO1)-independent oxidative stress and apoptosis induction.

The elevated expression of the flavoprotein NAD(P)H:quinone acceptor oxidoreductase (NQO1) (EC 1.6.99.2) in many human solid tumors, along with its ability to activate quinone-based anticancer agents, makes it an excellent target for enzyme-directed drug development. Previous studies have shown a significant statistical correlation between NQO1 enzymatic activity and the cytotoxicity of certain antitumor quinones. RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], presently in late preclinical and entering early clinical development, has been previously considered to be an excellent substrate for activation by NQO1. In this study we investigate the cytotoxicity of RH1 in cell lines selected from the NCI's 60 tumor cell line panel, expressing varying levels of NQO1 activity. Exposure time- and concentration-dependent cytotoxicity was seen, apparently independent from levels of NQO1 activity in these cells. Furthermore, the NQO1 inhibitor dicoumarol had no impact on the sensitivity profiles of RH1 response. The HL-60 myeloid leukemia cells, which do not have detectable NQO1 activity, were further investigated. RH1 treatment of HL-60 cells generated high levels of free radicals, which was accompanied by robust redox cycling, oxygen consumption and induction of apoptosis. These results are in agreement with previous data suggesting that, in addition to its activation by NQO1, RH1-induced cytotoxicity might involve alternative pathways for activation of this compound. Furthermore, the high cytotoxicity of RH1 in the leukemia/lymphoma subpanel of the NCI in vitro cell line screen would suggest an empirical rationale for the utilization of this compound in the treatment of these malignancies.