Multidrug-resistant cancer cells are preferential targets of the new antineoplastic lanthanum compound KP772 (FFC24).

Recently, we have introduced [tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772, FFC24) as a new lanthanum compound which has promising anticancer properties in vivo and in vitro. Aim of this study was to investigate the impact of ABC transporter-mediated multidrug resistance (MDR) on the anticancer activity of KP772. Here, we demonstrate that all MDR cell models investigated, overexpressing ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance protein 1), or ABCG2 (breast cancer resistance protein) either due to drug selection or gene transfection, were significantly hypersensitive against KP772. Using ABCB1-overexpressing KBC-1 cells as MDR model, KP772 hypersensitivity was demonstrated to be based on stronger apoptosis induction and/or cell cycle arrest at unaltered cellular drug accumulation. KP772 did neither stimulate ABCB1 ATPase activity nor alter rhodamine 123 accumulation arguing against a direct interaction with ABCB1. Accordingly, several drug resistance modulators did not sensitize but rather protect MDR cells against KP772-induced cytotoxicity. Moreover, long-term KP772 treatment of KBC-1 cells at subtoxic concentrations led within 20 passages to a complete loss of drug resistance based on blocked MDR1 gene expression. When exposing parental KB-3-1 cells to subtoxic, stepwise increasing KP772 concentrations, we observed, in contrast to several other metallo-drugs, no acquisition of KP772 resistance. Summarizing, our data demonstrate that KP772 is hyperactive in MDR cells and might have chemosensitizing properties by blocking ABCB1 expression. Together with the disability of tumor cells to acquire KP772 resistance, our data suggest that KP772 should be especially active against notoriously drug-resistant tumor types and as second line treatment after standard chemotherapy failure.

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.

The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts.

The ubiquitin protein ligase SCF(Skp2) is composed of Skp1, Cul1, Roc1/Rbx1 and the F-box protein Skp2, the substrate-recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re-enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen-independent S-phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G(0)/G(1) phases. Here we show that Skp2 is degraded by the proteasome in G(0)/G(1) and is stabilized when cells re-enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum-deprived cells induces Skp2 expression. Furthermore, recombinant Cul1-Roc1/Rbx1-Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G(0)/G(1) is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCF(Skp2) ubiquitin protein ligase function during the G(0)/G(1) phases of the cell cycle.

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells.

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells.

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin-protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway.

In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.