PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response.

BACKGROUND: Recent studies indicate that angiogenesis is important in the pathogenesis of acute myeloid leukemias (AMLs). Among the various AMLs, the bone marrow angiogenetic response is particularly pronounced in acute promyelocytic leukemia (APL). However, the molecular mechanisms responsible for this angiogenetic response are largely unknown. In the present study, we have explored the role of HHEX, a homeodomain transcription factor, as a possible mediator of the pro-angiogenetic response observed in APL. This transcription factor seems to represent an ideal candidate for this biologic function because it is targeted by PML-RARα, is capable of interaction with PML and PML-RARα, and acts as a regulator of the angiogenetic response. METHODS: We used various cellular systems of APL, including primary APL cells and leukemic cells engineered to express PML-RARα, to explore the role of the PML-RARα fusion protein on HHEX expression. Molecular and biochemical techniques have been used to investigate the mechanisms through which PML-RARα downmodulates HHEX and the functional consequences of this downmodulation at the level of the expression of various angiogenetic genes, cell proliferation and differentiation. RESULTS: Our results show that HHEX expression is clearly downmodulated in APL and that this effect is directly mediated by a repressive targeting of the HHEX gene promoter by PML-RARα. Studies carried out in primary APL cells and in a cell line model of APL with inducible PML-RARα expression directly support the view that this fusion protein through HHEX downmodulation stimulates the expression of various genes involved in angiogenesis and inhibits cell differentiation. CONCLUSIONS: Our data suggest that HHEX downmodulation by PML-RARα is a key event during APL pathogenesis.

Autocrine role of angiopoietins during megakaryocytic differentiation.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34(+) cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.

CDDO-Im is a stimulator of megakaryocytic differentiation.

Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.

Primary ovarian cancer cells are sensitive to the proaptotic effects of proteasome inhibitors.

Resistance of tumors to cell death signals poses a complex clinical problem. In the present study, we have explored the capacity of proteasome inhibitors to induce cell death of ovarian cancer cells. We explored the sensitivity of primary ovarian cancer cells to a combination of bortezomib (also known as PS-341), a proteasome inhibitor and TRAIL, a death ligand, or mapatumumab or lexatumumab, TRAIL-R1 or TRAIL-R2 targeting agonist monoclonal antibodies, respectively. The results of our study showed that the large majority of primary ovarian cancers are clearly sensitive to the pro-apoptotic action of bortezomib, whose effects are potentiated by the concomitant addition of TRAIL or mapatumumab or lexatumumab. Interestingly, both cisplatin and paclitaxel-chemosensitive and chemoresistant ovarian tumors are equally sensitive to the cytotoxic effect of bortezomib. Bortezomib, combined with TRAIL or TRAIL-R1 or TRAIL-R2 agonist monoclonal antibodies may be a useful treatment for refractory ovarian cancer.

Triterpenoids as new promising anticancer drugs.

Triterpenoids are structurally diverse organic compounds, characterized by a basic backbone modified in multiple ways, allowing the formation of more than 20 000 naturally occurring triterpenoid varieties. Several triterpenoids, including ursolic and oleanolic acid, betulinic acid, celastrol, pristimerin, lupeol, and avicins possess antitumor and anti-inflammatory properties. To improve antitumor activity, some synthetic triterpenoid derivatives have been synthesized, including cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic (CDDO), its methyl ester (CDDO-Me), and imidazolide (CDDO-Im) derivatives. Of these, CDDO, CDDO-Me, and betulinic acid have shown promising antitumor activities and are presently under evaluation in phase I studies. Triterpenoids are highly multifunctional and the antitumor activity of these compounds is measured by their ability to block nuclear factor-kappaB activation, induce apoptosis, inhibit signal transducer, and activate transcription and angiogenesis.

High sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide.

In the present study we have explored the sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/ADR and A2780/CISP, OVCAR3, SKOV3 and HEY cancer cell lines and primary ovarian cancer cells, providing evidence that: (i) the majority of these cell lines are highly sensitive to the pro-apoptotic effects induced by CDDO-Im; (ii) TRAIL, added alone exerted only a weak proapoptotic, but clearly potentiated the cytotoxic effect elicited by CDDO-Im; (iii) the apoptotic effect induced by CDDO-Im involves GSH depletion, c-FLIP downmodulation and caspase-8 activation; (iv) CDDO-Im inhibits STAT3 activation and CDDO-Im sensitivity is inversely related to the level of constitutive STAT3 activation. Importantly, studies on primary ovarian cancer cells have shown that these cells are sensitive to the pro-apoptotic effects of CDDO-Im. These observations support the experimental use of synthetic triterpenoids in the treatment of ovarian cancer.

A small molecule Smac mimic potentiates TRAIL-mediated cell death of ovarian cancer cells.

OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells. METHODS: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays. RESULTS: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells. CONCLUSIONS: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis.

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.