Characterization of the antiproliferative activity of Xylopia aethiopica.

BACKGROUND: Xylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells. RESULTS: We report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 µg/ml against HCT116 colon cancer cells, 7.5 µg/ml and > 25 µg/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid. CONCLUSION: The main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.

Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer.

BACKGROUND: The multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors. METHODS: In order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model. RESULTS: Compared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. CONCLUSIONS: These results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model.

The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

Cell cycle and apoptotic effects of SAHA are regulated by the cellular microenvironment in HCT116 multicellular tumour spheroids.

Using multicellular tumour spheroids (MCTS) of HCT116 colon carcinoma cells, we analysed the effects of SAHA (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor (HDACi). We found that, although SAHA-induced histone acetylation and ROS level upregulation occur throughout the spheroid, inhibition of cell cycle progression and induction of apoptosis are dependent on cell microenvironment. SAHA-induced growth inhibition of HCT116 MCTS results from the inhibition of cell cycle progression and induction of apoptosis. At a low concentration SAHA decreases Ki-67 and cyclin A positive cells and increases p21 positive cells in the outer layer while it induces a ROS-dependent apoptosis in the central zone of the spheroid. Coimmunostaining of p21 and apoptotic cells confirms that SAHA effects are different depending on the position of the cells within the spheroid. At a higher dose, SAHA induces mitotic defects and survivin downregulation in the outer layer of cells resulting in an additional cytotoxic effect in this part of the spheroid. Together these findings show that SAHA-induced cytostatic and cytotoxic effects occur in different cell populations, indicating that the cellular microenvironment is an important determinant in the regulation of the effects of SAHA treatment. Consequently, the MCTS model appears to be a valuable advanced tool for evaluating the effects of SAHA treatment in combination with other anticancer agents.

Benzo[e]pyridoindoles, novel inhibitors of the aurora kinases.

Aurora kinases are serine/threonine protein kinases that are involved in cancer development and are important targets for cancer therapy. By high throughput screening of a chemical library we found that benzo[e]pyridoindole derivatives inhibited Aurora kinase. The most potent compound (compound 1) was found to be an ATP competitive inhibitor, which inhibited in vitro Aurora kinases at the nanomolar range. It prevented, ex vivo, the phosphorylation of Histone H3, induced mitosis exit without chromosome segregation, known phenomena observed upon Aurora B inactivation. This compound was also shown to affect the localization of Aurora B, since in the presence of the inhibitor the enzyme was delocalized on the whole chromosomes and remained associated with the chromatin of newly formed nuclei. In addition, compound 1 inhibited the growth of different cell lines derived from different carcinoma. Its IC(50) for H358 NSCLC (Non Small Cancer Lung Cells), the most sensitive cell line, was 145 nM. Furthermore compound 1 was found to be efficient towards multicellular tumor spheroid growth. It exhibited minimal toxicity in mice while it had some potency towards aggressive NSCLC tumors. Benzo[e]pyridoindoles represent thus a potential new lead for the development of Aurora kinase inhibitors.

IRC-083864, a novel bis quinone inhibitor of CDC25 phosphatases active against human cancer cells.

CDC25 phosphatases are key actors in cyclin-dependent kinases activation whose role is essential at various stages of the cell cycle. CDC25 expression is upregulated in a number of human cancers. CDC25 phosphatases are therefore thought to represent promising novel targets in cancer therapy. Here, we report the identification and the characterization of IRC-083864, an original bis-quinone moiety that is a potent and selective inhibitor of CDC25 phosphatases in the low nanomolar range. IRC-083864 inhibits cell proliferation of a number of cell lines, regardless of their resistance to other drugs. It irreversibly inhibits cell proliferation and cell cycle progression and prevents entry into mitosis. In addition, it inhibits the growth of HCT-116 tumor spheroids with induction of p21 and apoptosis. Finally, IRC-083864 reduced tumor growth in mice with established human prostatic and pancreatic tumor xenografts. This study describes a novel compound, which merits further study as a potential anticancer agent.

Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents.

CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis. The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis. Here, we report that in p53(-/-) colon carcinoma cells, a moderate increase in the CDC25B level is sufficient to impair the DNA damage checkpoint, to increase spontaneous mutagenesis, and to sensitize cells to ionising radiation and genotoxic agents. Using a tumour cell spheroid assay as an alternative to animal studies, we demonstrate that the level of CDC25B expression modulates growth inhibition and apoptotic death. Since CDC25B overexpression has been observed in a significant number of human cancers, including colon carcinoma, and is often associated with high grade tumours and poor prognosis, our work suggests that the expression level of CDC25B might be a potential key parameter of the cellular response to cancer therapy.

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex.

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

OPA1 cleavage depends on decreased mitochondrial ATP level and bivalent metals.

OPA1, an intra-mitochondrial dynamin GTPase, is a key actor of outer and inner mitochondrial membrane dynamic. OPA1 amino-terminal cleavage by PARL and m-AAA proteases was recently proposed to participate to the mitochondrial network dynamic in a DeltaPsi(m)-dependent way, and to apoptosis. Here, by an in vitro approach combining the use of purified mitochondrial fractions and mitochondrial targeting drugs, we intended to identify the central stimulus responsible for OPA1 cleavage. We confirm that apoptosis induction and PTPore opening, as well as DeltaPsi(m) dissipation induce OPA1 cleavage. Nevertheless, our experiments evidenced that decreased mitochondrial ATP levels, either generated by apoptosis induction, DeltaPsi(m) dissipation or inhibition of ATP synthase, is the common and crucial stimulus that controls OPA1 processing. In addition, we report that ectopic iron addition activates OPA1 cleavage, whereas zinc inhibits this process. These results suggest that the ATP-dependent OPA1 processing plays a central role in correlating the energetic metabolism to mitochondrial dynamic and might be involved in the pathophysiology of diseases associated to excess of iron or depletion of zinc and ATP.

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

JNK is associated with Bcl-2 and PP1 in mitochondria: paclitaxel induces its activation and its association with the phosphorylated form of Bcl-2.

It has been shown that the activation of JNK after paclitaxel-induced microtubule damage is parallel to Bcl-2 phosphorylation, cell cycle arrest in mitosis and apoptosis. Using subcellular fractionation and immunocytochemistry, we found here that a pool of activated JNK is located in mitochondria of HeLa cells treated with paclitaxel. Furthermore, whereas the JNK protein is present in a tripartite complex with the anti-apoptotic Bcl-2 protein and the PP1 phosphatase in mitochondria isolated from control cells, the activated form of JNK was associated with the phosphorylated form of Bcl-2, but devoid of PP1, in mitochondria isolated from paclitaxel-treated cells. Moreover, using an original cell-free system, we evidenced a direct involvement of JNK as the kinase responsible for the phosphorylation of mitochondrial Bcl-2 in mitotic arrested cells. Indeed, cytosols prepared from mitotic arrested cells led to a dose-dependent phosphorylation of mitochondrial Bcl-2. Bcl-2 phosphorylation was inhibited by CEP 11004, a JNK pathway inhibitor and by immunodepletion of JNK. Taken together, these data show that JNK activation provides a molecular linkage from microtubule damages to the mitochondrial apoptotic machinery and also point to a pivotal role for the JNK/Bcl-2/PP1 complex in the control of apoptosis following paclitaxel treatment.

Paclitaxel increases p21 synthesis and accumulation of its AKT-phosphorylated form in the cytoplasm of cancer cells.

CKI p21 is a regulator of cellular responses to microtubule damage induced by drugs such as paclitaxel (PTX). It mediates the G1 4N arrest postactivation of the spindle assembly checkpoint and protects cancer cells against PTX-induced cytotoxicity. We demonstrated here that low doses of PTX that are unable to activate the spindle assembly checkpoint, upregulate p21 by a p53-dependent pathway and induce its translocation to the cytoplasm. This cytoplasmic accumulation of p21 resulted from an AKT-dependent p21 phosphorylation leading to an association of p21 with 14-3-3. Furthermore, the cytoplasmic p21 accumulation observed in PTX-treated cells was inhibited by LY 294002, a specific PI-3 kinase inhibitor or by the expression of a dominant-negative AKT mutant. However, the kinase activity of AKT was unchanged in PTX-treated cells, suggesting that low doses of PTX could regulate p21 phosphorylation via inhibition of its dephosphorylation. As a functional consequence, we found that cytoplasmic accumulation of the phosphorylated form of p21 prevents the inhibitory effect of p21, enabling these cells to escape to the p53-dependent Gl/S and G2/M checkpoints.

Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis.

OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae. These events are followed by cytochrome c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces mitochondrial fragmentation and apoptosis, the latter being inhibited by Bcl2 overexpression. These results suggest that OPA1 is a major organizer of the mitochondrial inner membrane from which the maintenance of the cristae integrity depends. As loss of OPA1 commits cells to apoptosis without any other stimulus, we propose that OPA1 is involved in the cytochrome c sequestration and might be a target for mitochondrial apoptotic effectors. Our results also suggest that abnormal apoptosis is a possible pathophysiological process leading to the retinal ganglion cells degeneration in ADOA patients.

Z-ajoene induces apoptosis of HL-60 cells: involvement of Bcl-2 cleavage.

Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.

The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space.

Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy. OPA1 encodes a dynamin-related protein orthologous to Msp1 of Schizosaccharomyces pombe and Mgm1p of Saccharomyces cerevisiae, both involved in mitochondrial morphology and genome maintenance. We present immuno-fluorescence and biochemical evidences showing that OPA1 resides in the mitochondria where it is imported through its highly basic amino-terminal extension. Proteolysis experiments indicate that OPA1 is present in the inter-membrane space and electron microscopy further localizes it close to the cristae. The strong association of OPA1 with membranes suggests its anchoring to the inner membrane.

PP1 phosphatase is involved in Bcl-2 dephosphorylation after prolonged mitotic arrest induced by paclitaxel.

During mitotic arrest induced by paclitaxel, most of the mitochondrial Bcl-2 is phosphorylated. This mitotic arrest is transient; exit from mitosis, due to mitotic slippage, occurs and Bcl-2 is rapidly dephosphorylated. In the present study, we characterized PP1 as the cytosolic phosphatase involved in Bcl-2 dephosphorylation. When mitochondria and cytosol prepared from mitotic arrested cells were incubated in vitro, the proportion of phosphorylated forms of Bcl-2 in mitochondria remained unchanged. In contrast, cytosol prepared from cells during mitotic slippage led to a dose-dependent loss of phosphorylated forms of Bcl-2. Depletion of these cytosol extracts by microcystin-Sepharose maintained Bcl-2 phosphorylated forms, indicating that this cytosol possessed phosphatase activity. Furthermore, the dephosphorylation of Bcl-2 by cytosol prepared from cells exiting mitotic block was inhibited by okadaic acid, at a dose known to inhibit PP1, and by inhibitor 2, a specific inhibitor of PP1 and by immunodepletion of PP1. Finally, we showed that PP1 is associated with mitochondrial Bcl-2 in vivo. Taken together, these results demonstrate that PP1 is directly involved in Bcl-2 dephosphorylation during mitotic slippage.

All trans retinoic acid enhances CDDP-induced apoptosis: modulation of the CDDP effect on cell cycle progression.

We have previously shown that ATRA potentiates CDDP cytotoxicity in various ovarian carcinoma cell lines. In the present study, we found that the enhanced sensitivity to CDDP was due to an increase of CDDP-induced apoptosis in OVCCR1 and NIH-OVCAR-3 cells. In these cell lines, flow cytometric analysis indicated that CDDP induced an initial accumulation of cells in the S-phase, followed by an increase in the proportion of cells in G2/M phase. Pretreatment of OVCCR1 and NIH-OVCAR-3 cells with ATRA did not modify cell cycle parameters, but delayed S-phase exit of CDDP-treated cells. Bcl-2 over-expression inhibited both delay in S-phase exit and CDDP-induced apoptosis in ATRA-pretreated cells. The CDDP-induced S-phase accumulation of OVCCR1 cells resulted from an activation of CDK2/cyclin A activity. Our results indicate that ATRA-pretreatment modified the CDDP-induced regulation of CDK2 activity by the CDK inhibitors p21 and p27. Taken together, our findings suggest that ATRA potentiates the apoptosis induced by CDDP in ovarian carcinoma cells and that this action is sustained by modulation of the activity of CDK2/cyclin A.