Juan Francisco Santibáñez

Structural Antitumoral Activity Relationships of Synthetic Chalcones

Relationships between the structural characteristic of synthetic chalcones and their antitumoral activity were studied. Treatment of HepG2 cells for 24 h with synthetic 2’-hydroxychalcones resulted in apoptosis induction and dose-dependent inhibition of cell proliferation. The calculated reactivity indexes and the adiabatic electron affinities using the DFT method including solvent effects, suggest a structure-activity relationship between the Chalcones structure and the apoptosis in HepG2 cells. The absence of methoxy substituents in the B ring of synthetic 2’-hydroxychalcones, showed the mayor structure-activity pattern along the series.

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2‐dependent TGF‐β signaling

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF‐β) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium‐derived nitric oxide in endoglin+/− (Eng+/−) mice and cultured endothelial cells. Here, we find that TGF‐β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/− mice, suggesting the involvement of endoglin in the TGF‐β regulated vascular homeostasis. The endoglin‐dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF‐β receptor ALK5 and its downstream substrate Smad2. In addition, Smad2‐specific signaling is upregulated in endoglin‐induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin‐dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin‐inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome‐dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF‐β signaling, and leading to an increased eNOS expression in endothelial cells. J. Cell. Physiol. 210: 456–468, 2007.

Transforming growth factor-β1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.

JNK mediates TGF-β1-induced epithelial mesenchymal transdifferentiation of mouse transformed keratinocytes

In this study we analyzed the role of the c‐Jun N‐terminal kinases (JNK) pathway in the TGF‐β1 stimulation of urokinase‐type plasminogen activator (uPA), initial stages of epithelial‐mesenchymal transdifferentiation (EMT) and cell migration. TGF‐β1 induces JNK phosphorylation, c‐Jun transactivation and AP1 activation. The involvement of JNK was evaluated using dominant negative mutants SEK‐1 AL, JNK and cJun, depletion of JNK1,2 proteins by treatment of cells with antisense oligonucleotides, as well as the chemical inhibitor SP600125. Our results demonstrated that the JNK pathway is required in the TGF‐β1 enhancement of uPA, fibronectin, E‐cadherin delocalization, actin re‐organization and vimentin expression, concomitant with the induction of cell migration. These results allow us to suggest a role of JNK in the TGF‐β1 induction of EMT in relation with the stimulation of malignant properties of mouse transformed keratinocytes.

The role of the TGF-β coreceptor endoglin in cancer

Endoglin (CD105) is an auxiliary membrane receptor of transforming growth factor beta (TGF-β) that interacts with type I and type II TGF-β receptors and modulates TGF-β signaling. Endoglin is overexpressed in the tumor-associated vascular endothelium, where it modulates angiogenesis. This feature makes endoglin a promising target for antiangiogenic cancer therapy. In addition, recent studies on human and experimental models of carcinogenesis point to an important tumor cell–autonomous role of endoglin by regulating proliferation, migration, invasion, and metastasis. These studies suggest that endoglin behaves as a suppressor of malignancy in experimental and human epithelial carcinogenesis, although it can also promote metastasis in other types of cancer. In this review, we evaluate the implication of endoglin in tumor development underlying studies developed in our laboratories in recent years.

ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase‐like plasminogen activator (u‐PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u‐PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u‐PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u‐PAR in this proliferative phenomenon. Our results show that in vitro u‐PA stimulates proliferation of SaOS‐2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS‐2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u‐PAR, and that culturing SaOS‐2 cells in the presence of u‐PA represents a stimuli for u‐PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate‐derived u‐PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u‐PA receptor.

Transforming Growth Factor-Beta and Oxidative Stress Interplay: Implications in Tumorigenesis and Cancer Progression.

Transforming growth factor-beta (TGF-β) and oxidative stress/Reactive Oxygen Species (ROS) both have pivotal roles in health and disease. In this review we are analyzing the interplay between TGF-β and ROS in tumorigenesis and cancer progression. They have contradictory roles in cancer progression since both can have antitumor effects, through the induction of cell death, senescence and cell cycle arrest, and protumor effects by contributing to cancer cell spreading, proliferation, survival, and metastasis. TGF-β can control ROS production directly or by downregulating antioxidative systems. Meanwhile, ROS can influence TGF-β signaling and increase its expression as well as its activation from the latent complex. This way, both are building a strong interplay which can be taken as an advantage by cancer cells in order to increment their malignancy. In addition, both TGF-β and ROS are able to induce cell senescence, which in one way protects damaged cells from neoplastic transformation but also may collaborate in cancer progression. The mutual collaboration of TGF-β and ROS in tumorigenesis is highly complex, and, due to their differential roles in tumor progression, careful consideration should be taken when thinking of combinatorial targeting in cancer therapies.

Genistein and Curcumin Block TGF-β1-Induced u-PA Expression and Migratory and Invasive Phenotype in Mouse Epidermal Keratinocytes

Transforming growth factor-β1 (TGF-β1) stimulates migration/invasion of mouse transformed keratinocytes and increases urokinase (u-PA) expression/secretion. In this report, we analyzed the biological behavior of two naturally occurring inhibitors of protein tyrosine kinases, genistein and curcumin, that could abrogate the enhancement of u-PA levels induced by TGF-β1 in transformed keratinocytes. Our results showed that genistein and curcumin blocked this response in a dose-dependent manner and also inhibited the TGF-β1-induced synthesis of fibronectin, an early responsive gene to the growth factor. Both compounds also reduced TGF-β1-stimulated cell migration and invasiveness. These results suggest that a tyrosine kinase-signaling pathway should be involved in TGF-β1-mediated increased malignancy of transformed keratinocytes and that genistein and curcumin could play an important role in inhibiting tumor progression.

Endoglin Regulates Cyclooxygenase-2 Expression and Activity

The endoglin heterozygous (Eng+/−) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng+/− mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E2 were observed in the Eng+/− mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng+/− but not in Eng+/+ mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N&ohgr;-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng+/+ mice. N&ohgr;-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-β1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng+/− mice.

Urokinase expression and binding activity associated with the transforming growth factor β1-induced migratory and invasive phenotype of mouse epidermal keratinocytes

Transforming growth factor β1(TGF‐β1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF‐β1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial‐fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous‐spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF‐β1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI‐1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF‐β1‐induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF‐β1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas. J. Cell. Biochem. 74:61–73, 1999.