Nutrition and Cancer, 60(1), 120–130 Copyright © 2008, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635580701613772 Induction of Apoptosis by Lupeol and Mango Extract in Mouse Prostate and LNCaP Cells Sahdeo Prasad, Neetu Kalra, and Yogeshwer Shukla Proteomics Laboratory, Industrial Toxicology Research Centre, Lucknow, India Prostate cancer (PCA) is one of the most invasive malignancy and second leading cause of cancer related deaths in United States and some other countries. Long latency period makes PCA an ideal disease for pharmacologic or nutritional chemoprevention. Lupeol, a triterpene present in mango and other fruits, has shown to possess anticancer properties in in vivo and in vitro assays. Here, we recorded the apoptogenic activity in mouse prostate by lupeol and mango pulp extract (MPE). Testosterone was injected subcutaneously (5 mg/kg body weight) for 14 consecutive days to male Swiss albino mice. Lupeol/MPE supplementation resulted in arrest of prostate enlargement in testosterone-treated animals. In mouse prostate tissue, lupeol and MPE supplementation resulted in a significantly high percentage of apoptotic cells in the hypodiploid region. The induction of apoptosis in mouse prostate cells was pre- ceded by the loss of mitochondrial transmembrane potential and DNA laddering. In testosterone-induced mouse prostate, upregula- tion of antiapoptotic B-cell non-Hodgkin lymphoma-2 and down- regulation of proapoptotic Bcl-2-associated X protein and caspase- 3 were also recorded. We further observed apoptogenic activities of lupeol in an in vitro model using human prostate cancer cells [lymph node carcinoma of the prostate (LNCaP)]. The apopto- genic response of lupeol-induced changes in LNCaP cells can be summarized as early increase of reactive oxygen species followed by induction of mitochondrial pathway leading to cell death. Thus, the results of this study demonstrate that lupeol/MPE is effective in combating testosterone–induced changes in mouse prostate as well as causing apoptosis by modulating cell-growth regulators. INTRODUCTION Prostate cancer (PCA) is one of the most invasive malignancy in males of the United States and many other countries and is the second leading cause of cancer deaths in American men and the men of some other countries (1,2). It is estimated that average an- nual PCA incidence rates in the United States are 110.4 to 180.9, whereas in India, it ranged from 5.0 to 9.1, per 100,000 (3). Epi- demiological, experimental, and clinical data point toward an Submitted 27 January 2007; accepted in final form 19 April 2007. Address correspondence to Yogeshwer Shukla, Environmental Car- cinogenesis Division, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow 226001, India. Phone: (+91) 094151-58430. FAX: (+91) 522-2628227. E-mail: yogeshwer shukla@hotmail.com and Shukla y@rediffmail.com. important role of androgens in the development and progres- sion of this disease (4). An increasing incidence of the disease and failure of conventional therapy for advanced invasive PCA leads to the search for new approaches for management of the disease (5,6). Epidemiological evidences have shown an in- verse association between consumption of vegetables and risk of PCA (7,8). Mango (Mangifera indica L.) is a worldwide con- sumed fruit in the tropical countries around the world. Mango extract has been shown to be antimutagenic in Salmonella typhimurium TA1538 (9). Chemical analysis of mango pulp extract (MPE) has shown that it contains vitamins, organic acids, carbohydrates, amino acids, polyphenols, and volatile compounds (10). Lupeol [Lup-20 (29)-en-3-β -ol; Fig. 1] is a naturally occurring pentacyclic triterpene present predom- inantly in mango. Lupeol has been shown to exhibit strong anti-inflammatory, antiarthritic, antimutagenic, and antimalarial activities (11–13). It has also been shown to possess antitumor- promoting effects in mouse skin carcinogenesis (14). The oral administration of lupeol changed the tissue redox system in- duced by cadmium exposure by scavenging the free radicals and by improving the antioxidant status of the rat liver (15). Generally, cancer is a multifactorial disease, which requires modulation of multiple pathways and targets. These targets in- clude the activation of apoptosis, suppression of growth factor expression, or signaling and downregulation of antiapoptotic proteins (16). Apoptosis is a well organized cell death process characterized by loss of plasma membrane phospholipid asym- metry, enzymatic cleavage of the DNA into oligonucleosomal fragments, and segmentation of the cells into membrane-bound apoptotic bodies (17). Genetic changes leading to loss of apop- tosis or derangement of apoptosis-signaling pathways in the transformed cells are critical for neoplastic changes (18). The execution process of cells by apoptosis is mediated by caspase- 3, one of a family of cysteine proteases (19,20). Activation of caspase-3 is tightly regulated by the formation of apoptosome (21), which is dependent on the cytochrome c release from the mitochondria into cytosol, which is thought to be regulated by B-cell non-Hodgkin lymphoma-2 (Bcl-2)-associated X protein (Bax) and Bcl-2. Proapoptotic Bax forms pores in the outer mitochondrial membrane, releasing cytochrome c, whereas an- tiapototic Bcl-2 prevents the opening of mitochondrial transition 120