Natural Triterpenic Diols Promote Apoptosis in Astrocytoma Cells through ROS-Mediated Mitochondrial Depolarization and JNK Activation

et al. (2009) Natural Triterpenic Diols Promote Apoptosis in Astrocytoma Cells through ROS-Mediated Mitochondrial Depolarization and JNK Activation. PLoS ONE 4(6): e5975. doi:10.1371/journal.pone.0005975 Natural Triterpenic Diols Promote Apoptosis in Astrocytoma Cells through ROS-Mediated Mitochondrial Depolarization and JNK Activation Rube n Martn 0 Elvira Ibeas 0 Juliana Carvalho-Tavares 0 Marita Herna ndez 0 Valentina Ruiz-Gutierrez 0 Mara Luisa Nieto 0 Joseph Alan Bauer, Bauer Research Foundation, United States of America 0 1 Instituto de Biolog a y Gene tica Molecular, CSIC-Universidad de Valladolid , Valladolid, Spain, 2 Instituto de la Grasa de Sevilla (CSIC), Sevilla , Spain Background: Triterpene alcohols and acids are multifunctional compounds widely distributed throughout the plant kingdom that exhibit a variety of beneficial health properties, being synthetic analogs of oleanolic acid under clinical evaluation as anti-tumoral therapeutic agents. However, the antineoplastic activity of two natural occuring triterpenoid alcohols extracted from olive oil, erythrodiol (an intermediate from oleanolic acid), and its isomer, uvaol, has barely been reported, particularly on brain cancer cells. Astrocytomas are among the most common and aggressive type of primary malignant tumors in the neurological system lacking effective treatments, and in this study, we addressed the effect of these two triterpenic diols on the human 1321N1 astrocytoma cell line. Principal Findings: Erythrodiol and uvaol effectively affected cell proliferation, as well as cell cycle phases and induced 1321N1 cell death. Both triterpenes successfully modulated the apoptotic response, promoting nuclear condensation and fragmentation. They caused retraction and rounding of cultured cells, which lost adherence from their supports, while Factin and vimentin filaments disappeared as an organized cytoplasmic network. At molecular level, changes in the expression of surface proteins associated with adhesion or death processes were also observed. Moreover, triterpene exposure resulted in the production of reactive oxygen species (ROS) with loss of mitochondrial transmembrane potential, and correlated with the activation of c-Jun N-terminal kinases (JNK). The presence of catalase reversed the triterpenic diolsinduced mitochondrial depolarization, JNK activation, and apoptotic death, indicating the critical role of ROS in the action of these compounds. Conclusions: Overall, we provide a significant insight into the anticarcinogenic action of erythrodiol and uvaol that may have a potential in prevention and treatment of brain tumors and other cancers. - Funding: This study has been funded by a grants SAF2005-01242, SAF2008-00245 and AGL2008-022845 from MICINN, and CSI11A08 from the Government of Castilla y Leon. MH is under the Ramon y Cajal Program (Co-funded by F.S.E.). E.I., R.M. and J.C-T. are supported by a fellowship from the Spanish Ministerio de Educacio n y Ciencia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Improvements in current cancer treatment regimens have resulted in an increase in patient survival. Nevertheless, many tumors, particularly astrogliomas, the most common primary brain tumors, relapse and are resistant to subsequent treatments. Several obstacles prevent the complete eradication of these highgrade malignant neoplasms by conventional therapies: i) their ability to infiltrate the surrounding normal brain, rendering total surgical excision highly improbable [1], ii) their low response or resistance to chemotherapy, and specially iii) their physical isolation from systemic circulation due to the impermeability of the bloodbrain barrier (BBB), which limits transport of most hidrophilic and large lipophilic molecules, thus preventing more than 95% of drugs from penetrating the brain [2]. Therefore, search new drugs or how to get these drugs across the natural guardian of the brain, the BBB, is the cutting edge research to find solutions to these highly invasive tumors. Accumulating data indicate that the cytotoxic effect of many chemotherapeutic drugs occurs through programmed cell death (apoptosis) [3]. Hence, the ability of tumor cells to respond and activate the apoptotic program may, in part, determine the success of the therapeutic strategy [4]. It is well documented that apoptosis can be induced by a variety of drugs with diverse chemical structure and different mechanism of action; and two major routes including the death-receptor pathway and the mitochondrialpathway have been identified [5]. Apoptosis is a highly regulated process that involves many proteins and genes [6,7]. It is characterized by cell shrinkage, plasma membrane bebbling, and chromatin condensation. The death program is executed by caspases, which amplify the apoptotic signal and proteolytically process numerous cellular molecules with different functions [8]. In response to death stimuli, ROS accumulation and alterations in the mitochondrial membrane potential (DYm) are considered to be early events [9]. In addition, mitogen-activated protein kinases (MAPK) have also been considered as an upstream signal for the initiation of apoptosis. Many studies have shown that the stressactivated protein kinases pathway are rapidly activated in response to oxidative insults and are frequently associated with cell death. Thus, their activation is usually correlated with apoptosis induced by agents that act, at least in part, via ROS generation [1013]. A critical genetic defect in many tumors, including gliomas, is found in the p53 gene, which makes p53 non-functional. Normally, p53 sensitizes the tumor cell to chemotherapy, which will induce programmed cell death. Thus, the search for effective agents to treat a broad spectrum of tumors that differ in the expression levels or in the mutational status of p53, are objectives of drug development programs. At present, it has been reported that members of the natural occurring triterpene family such as boswellic, maslinic, ursolic or betulinic acid among others, are potent apoptotic agents to cancer cells regardless of their p53 status (null, wild-type or mutant) [14,15]. Triterpenes are compounds widely available in fruits and vegetables in human diet, as in the components of natural herbal preparations used for the treatment of human diseases. Chemically pentacyclic triterpenes are all based on a 30-carbon skeleton comprising five six-membered rings (ursanes and oleananes) or four six-membered rings and one five-membered ring (lupanes), and as lipophilic molecules may also penetrate the blood-brain barrier, as it has already been demonstrated for some of them. [1618]. The plant Olea europaea, the origin of the cultivated olive, is widespread in Mediterranean countries, and extracts of its leaves, flow (...truncated)