Silibinin Strongly Synergizes Human Prostate Carcinoma DU145 Cells to Doxorubicin-induced Growth Inhibition, G 2 -M Arrest, and Apoptosis 1 Anil K. Tyagi, Rana P. Singh, Chapla Agarwal, Daniel C. F. Chan, and Rajesh Agarwal 2 Department of Pharmaceutical Sciences, School of Pharmacy [A. K. T., R. P. S., C. A., R. A.], Division of Medical Oncology, Department of Medicine [D. C. F. C.], and University of Colorado Cancer Center [D. C. F. C., R. A.], University of Colorado Health Sciences Center, Denver, Colorado 80262 ABSTRACT Purpose: We recently demonstrated the strong antican- cer efficacy of silibinin, an active constituent of a widely consumed dietary supplement milk thistle extract, against human prostate cancer cells in culture and nude mice xeno- grafts. We also observed that pharmacologically achievable concentrations of silibinin in animal studies were in the range of 25–100 M, depending on the dose regimen, which did not show any apparent toxicity to the animals. In this study, we assessed whether silibinin synergizes the therapeu- tic potential of the chemotherapeutic drug doxorubicin against prostate cancer, the effectiveness of which is limited because of high systemic toxicity. Experimental Design: Prostate cancer cells were treated with silibinin and doxorubicin, either alone or in combina- tion, and cell growth was determined by manual cell count- ing. Cell cycle progression was assessed by saponin/ propidium iodide staining and fluorescence-activated cell sorter analysis. Protein levels of cell cycle regulators were determined by Western blotting, and cdc2/p34 kinase activ- ity was analyzed by in-beads kinase assay. Apoptosis was quantified by annexin V/propidium iodide staining and fluorescence-activated cell sorter analysis. Results: Silibinin strongly synergized the growth-inhib- itory effect of doxorubicin in prostate carcinoma DU145 cells (combination index, 0.235– 0.587), which was associated with a strong G 2 -M arrest in cell cycle progression, showing 88% cells in G 2 -M phase by this combination compared with 19 and 41% of cells in silibinin and doxorubicin treatment alone, respectively. The underlying mechanism of G 2 -M ar- rest showed a strong inhibitory effect of combination on cdc25C, cdc2/p34, and cyclin B1 protein expression and cdc2/p34 kinase activity. More importantly, this combina- tion caused 41% apoptotic cell death compared with 15% by either agent alone. Silibinin and doxorubicin alone as well as in combination were also effective in inhibiting the growth of androgen-dependent prostate carcinoma LNCaP cells. Conclusion: These findings suggest a need for in vivo studies with this combination in preclinical prostate cancer models. Positive outcomes might be relevant for a clinical application in prostate cancer patients. INTRODUCTION PCA 3 is the leading cause of death from cancer in older men and the most commonly diagnosed cancer in men overall (1). For confined disease of low to moderately differentiated PCA, surgical or radiotherapy techniques are curative in the majority of individuals (2, 3). Unfortunately, there is no effec- tive cure for PCA once the cancer has spread beyond the pelvis; these patients are estimated to account for 30,200 deaths in 2002 (1). Cytotoxic chemotherapy is being used to control and treat PCA at this stage but remains relatively nonselective and highly toxic to normal tissues. Doxorubicin is one of the clinical chemotherapy agents, which possesses a broad spectrum of therapeutic activity against various cancers including PCA (4, 5). However, the success of doxorubicin chemotherapy in PCA patient is limited, because its sufficient concentration could not be achieved without systemic toxicity (4). The reduction in doxorubicin-caused systemic toxicity poses a major challenge in maximizing the beneficial outcome of its therapy in PCA pa- tients. Because of these limitations, there has been intense public and scientific interest in the use of other approaches to control the growth of PCA as well as its treatment. In an effort to develop effective strategies that increase the therapeutic po- tential of cytotoxic anticancer drugs with less systemic toxicity in recent years, more efforts are being directed toward combi- nation chemotherapy (6, 7). In this regard, dietary supplements as well as phytotherapeutic agents with high anticancer efficacy and least toxicity to normal tissues are suggested as possible candidates to be investigated for their synergistic efficacy in combination with anticancer drugs (8 –11). In recent studies, we have reported cancer chemopreven- tive and therapeutic efficacy of silibinin, an antioxidant fla- vonoid, present in a widely consumed dietary supplement milk Received 4/18/02; revised 6/27/02; accepted 6/28/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by USPHS Grants CA83741 and CA64514 (to R. A.). In part, the work reported here was performed when the authors (A. K. T., C. A., and R. A.) were at American Medical Center Cancer Research Center, Denver, CO. 2 To whom requests for reprints should be addressed, at Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East Ninth Street, Box C238, Denver, CO 80262. Phone: (303) 315-1381; Fax: (303) 315-6281; E-mail: Rajesh.Agarwal@UCHSC.edu. 3 The abbreviations used are: PCA, prostate cancer; CI, combination index; PI, propidium iodide. 3512 Vol. 8, 3512–3519, November 2002 Clinical Cancer Research Research. on November 25, 2021. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from