Pandey1* Abstract Selective Cytotoxicity against Human Osteosarcoma Cells by a Novel Synthetic C-1 Analogue of 7- Deoxypancratistatin Is Potentiated by Curcumin Dennis Ma 1 , Phillip Tremblay1, Kevinjeet Mahngar1, Jonathan Collins2, Tomas Hudlicky2, Siyaram 1 Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada, 2 Chemistry Department and Centre for Biotechnology, Brock University, St. Catharines, Ontario, Canada The natural compound pancratistatin (PST) is a non-genotoxic inducer of apoptosis in a variety of cancers. It exhibits cancer selectivity as non-cancerous cells are markedly less sensitive to PST. Nonetheless, PST is not readily synthesized and is present in very low quantities in its natural source to be applied clinically. We have previously synthesized and evaluated several synthetic analogues of 7-deoxypancratistatin, and found that JC-TH-acetate-4 (JCTH-4), a C-1 acetoxymethyl analogue, possessed similar apoptosis inducing activity compared to PST. In this study, notoriously chemoresistant osteosarcoma (OS) cells (Saos-2, U-2 OS) were substantially susceptible to JCTH-4-induced apoptosis through mitochondrial targeting; JCTH-4 induced collapse of mitochondrial membrane potential (MMP), increased reactive oxygen species (ROS) production in isolated mitochondria, and caused release of apoptosis inducing factor (AIF) and endonuclease G (EndoG) from isolated mitochondria. Furthermore, JCTH-4 selectively induced autophagy in OS cells. Additionally, we investigated the combinatory effect of JCTH-4 with the natural compound curcumin (CC), a compound found in turmeric spice, previously shown to possess antiproliferative properties. CC alone had no observable effect on Saos-2 and U-2 OS cells. However, when present with JCTH-4, CC was able to enhance the cytotoxicity of JCTH-4 selectively in OS cells. Such cytotoxicity by JCTH-4 alone and in combination with CC was not observed in normal human osteoblasts (HOb) and normal human fetal fibroblasts (NFF). Therefore, this report illustrates a new window in combination therapy, utilizing a novel synthetic analogue of PST with the natural compound CC, for the treatment of OS. Citation: Ma D, Tremblay P, Mahngar K, Collins J, Hudlicky T, et al. (2011) Selective Cytotoxicity against Human Osteosarcoma Cells by a Novel Synthetic C-1 Analogue of 7-Deoxypancratistatin Is Potentiated by Curcumin. PLoS ONE 6(12): e28780. doi:10.1371/journal.pone.0028780 Editor: Regine Schneider-Stock, Erlangen University, Germany Received July 7, 2011; Accepted November 15, 2011; Published December 21, 2011 Copyright: 2011 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding of this work has been provided as a generous gift by the Knights of Columbus Chapter 9671 (Windsor, ON, Canada). This work has also been supported by a Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship awarded to Dennis Ma. 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 read the journal’s policy and have the following conflict: Dr. Pandey serves on the editorial board for PLoS ONE. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: spandey@uwindsor.ca Introduction For many centuries, a plethora of natural products have been used in traditional medicine for the treatment of numerous ailments. One such product includes the Curcuma longa herb [1]. Traditionally, this herb has been used to treat anorexia, rheumatism, sinusitis, hepatic disorders, and inflammation [2]. More recently, a component of this herb, the compound curcumin (CC) also referred to as (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphe- nyl)-1,6-heptadiene-3,5-dione or diferuloylmethane, has been recognized for its antiproliferative properties in treating cancer [1]. In particular, CC has been shown to regulate expression of genes implicated in cell proliferation, metastasis, chemotherapy resistance, and angiogenesis [1,3]. The anti-neoplastic properties of CC are exhibited in many types of malignancies including breast cancer, colon cancer, kidney cancer, leukemia, prostate cancer, melanoma, and osteosarcoma (OS) [1,4]. OS, a primary malignant bone tumor, is an extremely aggressive form of cancer associated with poor prognosis [5]. It occurs most commonly in the developing bones of children and adolescents and is often accompanied by lung metastases and subsequent respiratory failure [6,7]. Current treatment strategies include surgery and radiation therapy with adjuvant chemother- apy with agents such as doxorubicin, cisplatin, methotrexate, etoposide, and ifosfamide at high doses [6]. Despite the use of these chemotherapeutics, the 5-year survival rate for patients with metastatic OS is only 20% [6]. Furthermore, toxicity has been associated with the use of these drugs and chemoresistance frequently develops in this aggressive cancer; thus, more selective and effective chemotherapeutics are needed for OS [7–11]. Previously, we have shown the natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, to induce cytotoxicity in a number of malignant cell lines at concentrations below 1 mM and reduce the volume of human prostate and colon tumor xenografts [12–18]. Contrasting from many approved chemotherapeutics in use, non-cancerous cell lines are markedly less sensitive to PST [12–18]. PST however, is present in only parts per million quantities in its natural source and there have been many difficulties associated with its chemical synthesis; therefore, the major bottleneck of this compound has been its low PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28780