Tempranillo-derived grape seed extract induces apoptotic cell death and cell growth arrest in human promyelocytic leukemia HL-60 cells Javier Espino, * a David Gonz´ alez-G´ omez, bc Daniel Moreno, c Mar´ ıa F. Fern´ andez-Le´ on, c Ana B. Rodr´ ıguez, a Jos´ e A. Pariente a and Jonathan Delgado-Ad´ amez c Although grape seed extract (GSE) has proven to be effective against various cancers, few studies have investigated the effects of GSE on human leukemia. In this study, we analysed the mechanisms involved in the apoptotic effects induced by GSE on human promyelocytic leukemia HL-60 cells. Thus, GSE treatment succeeded in activating caspase-3 (P < 0.05), the activation being dose-dependent and time- dependent. Activation of caspase-3 induced by GSE was accompanied by mitochondrial membrane depolarization (P < 0.05). Moreover, disruption of mitochondrial integrity caused by GSE treatment subsequently led to activation of caspase-9 (P < 0.05), and also produced a slight increase in ROS levels (P < 0.05). Cytotoxic effects elicited by GSE treatment ultimately resulted in extensive S-phase arrest (P < 0.05) and a substantial increase in the intrinsic rate of apoptosis (P < 0.05). Our findings suggest that the GSE induces apoptotic cell death and cell growth inhibition in human leukemic HL-60 cells, which seems to be dependent on mitochondrial damage. Therefore, the GSE obtained from Tempranillo cultivars could be an effective approach to restrain uncontrolled cell proliferation and survival in leukemia cells. Introduction Cancer is a major cause of death and disability worldwide that affects both the physical and emotional well-being of individ- uals and their families, and ultimately poses an economic challenge to the community in terms of health care infrastruc- ture. Because the incidence of cancer varies dramatically from country to country, 1 much emphasis has been placed on understanding the environmental factors affecting cancer risk, including diet. Thus, epidemiological studies have suggested that developing healthy eating habits, e.g., regular consumption of fruits and vegetables, reduces cancer risk. 2 Based on these observations, a number of programs advocating the prevention of cancer encourage the consumption of at least ve portions of colorful fruits and vegetables each day. 3 Consequently, in recent years, cancer research has focused its attention on the isolation and characterization of potential chemopreventive agents present in fruits and vegetables. 4 Thus, certain dietary compo- nents, including vitamins, polyphenols and phytoestrogens, have already shown encouraging anticancer efficiency in different immortalized cell lines as well as animal xenogra models. 5 Grape (Vitis vinifera L.) is one of the fruit crops most widely grown throughout the world, 6 with Tempranillo cultivars being the variety most extensively grown in the Extremadura region, southwestern Spain. Grape seeds are considered a relevant source of phytochemicals. 7 Particularly, grape seed extract (GSE) offers promising new options for the development of more effective chemotherapeutic strategies against different types of cancers, such as breast, colorectal and lung cancer. 8–10 GSE is readily extracted from grape seeds, a by-product of grape juice and wine industries, and contains a mixture of several polyphenolic components, which constitute dimers, trimers, tetramers, and oligomers/polymers of monomeric catechins and/or epicatechins. 7 Most of the benecial effects of the GSE are attributed to their antioxidant and free radical scavenging properties. 11–13 Interestingly, it is now believed that at least some of the constituents present in the GSE act synergistically and may provide better anticancer effects than a single constituent. 10 Acute myeloid leukemia (AML) is the most common myeloid leukemia, with a prevalence of 3–8 cases per 100 000 that rises to 17–19 per 100 000 in those older than 65 years. It accounts for 30% of all cases of leukemia in adults, and 18 000 new patients are diagnosed in Europe each year, representing a Department of Physiology, Neuroimmunophysiology and Chrononutrition Research Group, Faculty of Science, University of Extremadura, 06006 Badajoz, Spain. E-mail: jespino@unex.es; Fax: +34 924289388; Tel: +34 924289388 b Department of Analytical Sciences, Faculty of Science, National University of Distance Education (UNED), Madrid, Spain c Technological Institute of Food and Agriculture (INTAEX), Gobierno de Extremadura, Badajoz, Spain Cite this: Food Funct., 2013, 4, 1759 Received 12th July 2013 Accepted 21st September 2013 DOI: 10.1039/c3fo60267b www.rsc.org/foodfunction This journal is ª The Royal Society of Chemistry 2013 Food Funct., 2013, 4, 1759–1766 | 1759 Food & Function PAPER Published on 24 September 2013. Downloaded by The University of Manchester Library on 22/10/2014 22:09:52. View Article Online View Journal | View Issue