Gerald Schmid

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1‐phosphate receptor 1

FTY720, a sphingosine 1‐phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti‐proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF‐A‐stimulated endothelial cells even at concentrations that apparently had no anti‐proliferative effect. Mechanistically, the anti‐angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti‐angiogenic effect of FTY720 was abrogated in the presence of CXCR4‐neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti‐angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel™ plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development. J. Cell. Biochem. 101: 259–270, 2007.

Dual action of the inhibitors of cyclin-dependent kinases: targeting of the cell-cycle progression and activation of wild-type p53 protein

The inhibition of cyclin-dependent kinases (CDKs) represents a novel approach to the therapy of human malignancies. Already in clinical trials, recently developed CDK inhibitors very efficiently target the rapidly proliferating cancer cells and inhibit their cell-cycle progression. Interestingly, some CDK inhibitors additionally affect the stability and activity of the tumour-suppressor protein p53, thereby enhancing their antiproliferative action towards cancer cells. Considering the fact that the p53 protein is mutated or inactivated in ~ 50% of all human cancers, the efficacy of CDK inhibitor therapy could differ between cancer cells depending on their p53 status. Moreover, recent reports demonstrating that some cancer cells can proliferate despite CDK2 inhibition questioned the central role of CDK2 in the cell-cycle control and suitability of CDK2 as a therapeutic target; however, the p53 activation that is mediated by CDK inhibitors could be essential for the efficacy of CDK inhibitors in therapy of CDK2-independent cancers. Furthermore, there is also reason to believe that CDK2 inhibitors could be used for another purpose, to protect normal cells from the effects of chemotherapy.

Inhibition of farnesyl protein transferase sensitizes human MCF‐7 breast cancer cells to roscovitine‐mediated cell cycle arrest

We reported recently that roscovitine (ROSC), a selective cyclin‐dependent kinase (CDK) inhibitor, arrests human MCF‐7 breast cancer cells in G2 phase of the cell cycle, and concomitantly induces apoptosis. Human MCF‐7 breast cancer cells are known to express elevated levels of c‐Ha‐Ras protein. To achieve full biological activity, de novo synthesized c‐Ha‐Ras protein has to be farnesylated and after further processing it needs to be attached to the plasma membrane. Therefore, we decided to prove whether prevention of protein farnesylation would sensitize MCF‐7 cells to the action of ROSC. MCF‐7 cells were treated with 1–40 µM ROSC alone, or in combination with L‐744,832, an inhibitor of farnesyl protein transferase (FTPase). To measure the impact on the proliferation of the cells, we used the CellTiterGlo™ viability assay and FACS analysis was employed to quantify the DNA‐content of the single cells. The amount and phosphorylation status of relevant proteins after lysis of MCF‐7 cells was assessed on Western blots using (phospho)‐specific antibodies. The combined treatment with L‐744,832 and ROSC for 24 h, markedly reduced the number of viable MCF‐7 cells, primarily, by re‐enforcing the cell cycle arrest. Interestingly, the potentiation of the ROSC‐mediated inhibition of cell proliferation became evident during the 48 h post‐incubation period in presence of the FPTase inhibitor. Inhibition of FPTase in ROSC‐treated cells reduced the number of viable cells by approximately 30%. Evidently, the combined treatment sensitizes MCF‐7 cells to the action of ROSC, thereby allowing to reduce the dose of the drug and to minimize side effects. J. Cell. Biochem. 102: 736–747, 2007.

Prevention of farnesylation of c‐Ha‐Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells

Ras, the product of a proto‐oncogene, is a GTP‐hydrolyzing enzyme found mutated in approximately 50% of human cancers. “Gain of function” mutations of Ras lead to an escape of transformed cells from cell‐cycle control, rendering them independent to stimulation by growth factors, giving them almost unlimited proliferation capacity. The cytosolic precursor isoform of Ras is biologically inactive. After several post‐translational modifications, Ras is anchored to the plasma membrane and, thereby, the protein becomes activated. The finding that lipid modifications of Ras protein, particularly farnesylation, are essential for its signal transduction activity, gave rise to the concept that blocking farnesyl protein transferase (FPTase), the enzyme catalyzing the first step in the Ras modification cascade, would prevent proper membrane anchoring and provide an improved approach in the cure of tumors harboring Ras mutations. In the present study we used transformed rat cells overexpressing a temperature‐sensitive p53 protein, adopting wt conformation at 32°C and mutant conformation at 37°C. We treated the cells growing at 32 or 37°C with doxorubicin alone, or in combination with inhibitors of FPTase. Combined treatment was more efficient and the same inhibition of cell proliferation was reached at lower DOX concentrations. The treatment strongly affected the growth rate of tumor cells but only negligibly of normal cells. However, the inhibitors of FPTase prevented the membrane anchoring in both situations. These results show two striking advantages of the combined treatment: the desired cytostatic effect on tumor cells at lower drug concentrations and clearly reduced adverse effects on quiescent cells. J. Cell. Biochem. 99: 1664–1676, 2006.