Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Anti-mitotic properties of indirubin-3'-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest.

The bis-indole indirubin is the active ingredient of the Traditional Chinese Medicine recipe Danggui Longhui Wan used against chronic myelocytic leukemia. We have previously shown that indirubins are potent inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3. We here investigated the anti-mitotic properties of this class of compounds using the cell permeable indirubin-3'-monoxime and the HBL-100 cell line. Indirubin-3'-monoxime reversibly arrests asynchronous HBL-100 cells in G2. This arrest is not accompanied by any significant change in expression of the major cell cycle regulators. However indirubin-3'-monoxime inhibits the phosphorylation of consensus CDK phosphorylation sites as well as of nucleolin at a specific CDK1/cyclin B phosphorylation site, suggesting a direct action on the mitotic CDK1/cyclin B. When indirubin-3'-monoxime is added to HBL-100 cells synchronized in M phase by nocodazole, cells undergo an endoreplication leading to an 8n DNA content. As soon as indirubin-3'-monoxime is washed away, these polyploid cells become aneuploid and later die from necrosis. This mechanism of endoreplication followed by cell death may contribute to the anti-tumour properties of indirubins.

Effects of human lactoferrin on NK cell cytotoxicity against haematopoietic and epithelial tumour cells.

Lactoferrin is an iron-binding glycoprotein implicated in particular in the control of immune functions and cell proliferation. We have investigated its involvement, at inflammatory concentrations, in cancer progression. We report that lactoferrin has a significant effect on natural killer (NK) cell cytotoxicity against haematopoietic and breast epithelial cell lines. Lactoferrin increases cytolysis at a low concentration (10 micrograms/ml) while at a high concentration (100 micrograms/ml) it modulates cytolysis depending on the target cell phenotype. By pre-treatment of either NK cells or target cells with lactoferrin, we have demonstrated that the lactoferrin effect is due both to a modulation of NK cell cytotoxicity and the target cell sensitivity to lysis. Lactoferrin binds to 91% of the naturally heterogeneous CD56dim/bright NK cell population and increases the NK cell cytotoxic activity at low concentrations. High concentrations of lactoferrin seem to be toxic for the CD56bright NK cells and decrease NK cell cytotoxicity. Lactoferrin also exerts an effect on target cells depending on the cell phenotype. It does not modify the susceptibility to lysis of haematopoietic cells such as Jurkat and K-562 cells, but does significantly increase that of the breast and colon epithelial cells. We have also demonstrated that lactoferrin inhibits epithelial cell proliferation by blocking the cell cycle progression.

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.

Role of heparan sulphate proteoglycans in the regulation of human lactoferrin binding and activity in the MDA-MB-231 breast cancer cell line.

We previously demonstrated that lactoferrin increases breast cell sensitivity to natural killer cell cytotoxicity whereas haematopoietic cells are unaffected by lactoferrin. It has been described that lactoferrin binds to various glycosaminoglycans. Compared to haematopoietic cells, breast cancer cells and particularly the breast cell line MDA-MB-231, possess a high level of proteoglycans. Scatchard analysis of 125I-lactoferrin binding to MDA-MB-231 cells revealed the presence of two classes of binding sites: a low affinity site with a Kd of about 700 nM and 3.9 x 10(6) sites and a higher affinity class with a Kd of 45 nM and 2.9 x 10(5) sites per cell. To investigate the potential regulation of lactoferrin activity by proteoglycans expressed on the MDA-MB-231 cells, we treated these cells with glycosaminoglycan-degrading enzymes or sodium chlorate, a metabolic inhibitor of proteoglycan sulphation. We showed that chondroitinase treatment has no effect, while heparinase or chlorate treatment significantly reduces both the binding of lactoferrin to cell surface sulphated molecules such as heparan sulphate proteoglycans (HSPG) and the affinity of lactoferrin for the higher affinity binding sites. The modulation of the lactoferrin binding was correlated with a decrease in lactoferrin activities on both MDA-MB-231 cell sensitisation to lysis and proliferation. Taken together, these results suggest that the presence of adequately sulphated molecules, in particular HSPG, is important for lactoferrin interaction and activity on the breast cancer cells MDA-MB-231.

[Prevention of chemotherapy-induced alopecia by cyclin-dependant kinase inhibitors]. / Prévention par un inhibiteur de kinases cycline-dépendantes, de l'alopécie induite par la chimiothérapie.

Cyclin-dependent kinases (CDKs) are widely involved in regulating the cell division cycle. The discovery of numerous alterations of CDKs, cyclins, their regulators and their substrates in many human tumours has strongly stimulated the search for chemical inhibitors of CDKs. The potential use of these potent and selective kinase inhibitors in cancer therapy is presently under investigation. Recently a group from Glaxo Wellcome unexpectedly discovered a potential new application of these CDK inhibitors to prevent chemotherapy-induced alopecia. These cyto-protective properties might also be extended to other tissues damaged during chemotherapy.

Molecular events that regulate cell proliferation: an approach for the development of new anticancer drugs.

Cancer chemotherapy is the object of many fundamental and clinical researches. The development in molecular techniques and structural studies at the molecular level have led to the discovery of key proteins involved in the regulation of cell proliferation. This opened perspectives to characterize new anticancer drugs in order to reduce the limitations found with conventional drugs such as the lack of selectivity for cancer cells and resistance phenomena. This review presents the anticancer drugs in clinical investigations that target molecules involved in the signal transduction impairment, the cell cycle deregulation and the differentiation with comments on their mechanisms of action.

[Chemical inhibitors of cyclic-dependent kinases: preclinical and clinical study]. / Inhibiteurs chimiques des kinases cyclines-dépendantes: ètudes précliniques et cliniques.

In the past decade, the use of a large variety of cellular models and cell biology, biochemistry and molecular biology techniques has led to the discovery of key proteins that are intimately involved in the regulation of tumor growth. In particular, it has been shown that cyclin-dependent kinases (CDKs) are key regulators of the cell-division cycle. Their frequent deregulation in human tumors make them attractive targets for the identification of new antineoplasic agents. Intensive screening has led in the past few years to the identification of a series of selective and potent chemical inhibitors of CDKs. Drugs representing new lead structures like flavopiridol, indirubin and staurosporine++ derivatives have already been used in clinical evaluation for cancer treatment (clinical trials, phase I and II). Anticancer drug development is being pursued to reduce their toxic side effects, to improve their pharmacokinetic properties and to increase their anti-tumor activity. In this context, traditional drug screening methods in biological test systems have led to the discovery of new compounds such as purine derivatives and paullones, which display remarkable selectivity and efficiency. These novels drugs may result in substantial progress in cancer treatment in the near future.

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.

Lactoferrin upregulates the expression of CD4 antigen through the stimulation of the mitogen-activated protein kinase in the human lymphoblastic T Jurkat cell line.

The main biological properties of lactoferrin are thought to concern inflammation and immunomodulation processes, including maturation of immature B and T cells. Lactoferrin accelerates T-cell maturation by inducing the expression of the CD4 surface marker. In this report, using the Jurkat T-cell line, we have shown that lactoferrin upregulates the expression of CD4 antigen through the activation of a transduction pathway. Using an anti-phosphotyrosine antibody, lactoferrin was demonstrated to induce a cascade of phosphorylation of numerous proteins on their tyrosine residues. This tyrosine-phosphorylation was transient, reaching maxima between 5 and 10 min. We also identified the mitogen-activated protein kinase (MAP kinase) which presented an enhanced catalytic activity, reaching a maximum at 10 min of incubation with lactoferrin. Moreover, the use of inhibitors such as genistein and PD98059, tyrosine kinases and MAP kinase kinase (or MEK) inhibitors respectively, allowed us to correlate the activation of MAP kinase with the upregulation of CD4 expression. Finally, using Lck-defective Jurkat cells, our results showed that the p56(lck) (Lck) kinase is necessary for MAP kinase activity and CD4 expression. This paper demonstrates that lactoferrin activates transduction pathway(s) in lymphoblastic T-cells, and that Lck and the Erk2 isoform of MAP kinase are implicated in the upregulation of CD4, induced by lactoferrin in these cells.

Coscinosulfate, a CDC25 phosphatase inhibitor from the sponge Coscinoderma mathewsi.

The dual specificity CDC25 phosphatases dephosphorylate two inhibitory phospho-amino acids of cyclin-dependent kinases, a major family of cell cycle regulators. CDC25 inhibitors constitute new anti-mitotic agents with potential anticancer activity. While screening through a collection of natural products derived from marine organisms for CDC25A inhibitors, we purified and identified coscinosulfate 1, a sesquiterpene sulfate from the New Caledonian sponge Coscinoderma matthewsi, along with 4. The purified compound 1 displayed significant inhibitory activity towards CDC25A (IC(50): 3 microM).