Diltiazem Enhances the Apoptotic Effects of Proteasome Inhibitors to Induce Prostate Cancer Cell Death Ismail Kaddour-Djebbar, Vivek Choudhary, Vijayabaskar Lakshmikanthan, 1 Robert Shirley, Manal El Gaish, Mohamed Al-Shabrawey, Belal Al-Husein, Roger Zhong, Michael Davis, Zheng Dong, Wendy B. Bollag, and M. Vijay Kumar 2 Charlie Norwood VA Medical Center, Augusta, Georgia (I.K.-D., V.C., V.L., R.S., M.E.G., B.A.-H., R.Z., M.D., Z.D., W.B.B., M.V.K.); Department of Surgery (Urology), (I.K.-D., V.C., V.L., R.S., M.V.K.); Departments of Physiology (I.K.-D., V.C., W.B.B.), Oral Biology (M.A.-S., W.B.B.), and Cell Biology and Anatomy, Georgia Health Sciences University, Augusta, Georgia (Z.D., W.B.B.); and College of Pharmacy, University of Georgia, Augusta, Georgia (B.A.-H.) Received September 17, 2011; accepted March 2, 2012 ABSTRACT Diltiazem is a calcium channel blocker used to treat cardiovascular ailments. In addition, reports suggest that diltiazem induces cell death, which could make it a drug of choice for the treatment of cancer associated with hypertension. The goal of this research was to determine whether diltiazem is capable of inducing apo- ptosis in prostate cancer cells, either alone or in combination with the proteasome inhibitors, lactacystin and bortezomib (Velcade). Bortezomib is approved for the treatment of multiple myeloma; unfortunately, it has side effects that limit its utility. Presumably these side effects could be decreased by reducing its dose in combination with another drug. We have previously shown that lactacystin induces apoptosis in LNCaP cells; here, we show that this effect was enhanced by diltiazem. Furthermore, in protea- some inhibitor-resistant DU145 cells, diltiazem alone did not in- duce apoptosis but decreased cytosolic calcium levels and in- duced mitochondrial fission; likewise, lactacystin did not induce apoptosis but up-regulated the proapoptotic protein Bik. How- ever, increasing concentrations of diltiazem in combination with lactacystin or bortezomib induced apoptosis in a dose-dependent and synergistic manner. The combination of diltiazem and lacta- cystin also up-regulated the levels of Bik and released Bak from Bcl-xL, indicating the involvement of the Bcl2 family pathway in this apoptosis. In addition, the drug combination up-regulated GRP78, suggesting also the involvement of endoplasmic reticu- lum stress in the apoptotic response. Thus, our results demon- strate a potential therapeutic advantage of combining a frequently used calcium channel blocker with proteasome inhibitors in the treatment of prostate cancer. Introduction The concentration of cytosolic Ca 2+ ([Ca 2+ ] c ) is maintained at a lower level inside the cell compared with that in the extracellular space. This gradient of Ca 2+ is tightly con- trolled by interactions with binding proteins and movement across the plasma membrane, in addition to transport into and out of key organelles such as the endoplasmic reticulum (ER) and mitochondria. The ER plays the role of a calcium reservoir, whereas mitochondria are a calcium sink, remov- ing and buffering excess calcium in the cytosol (Ferreiro et al., 2008). Finely tuned changes in [Ca 2+ ] c modulate a vari- ety of intracellular functions ranging from muscle contrac- tion to secretion. Ionic calcium also plays a major role in the complex interplay that leads to cell death (Tagliarino et al., 2001; Roderick and Cook, 2008). Indeed, Ca 2+ signals in the nucleus, ER, and mitochondria have been shown to affect checkpoints of the cell death process, thus modulating the sensitivity of cells to various challenges (Gill et al., 1996). [Ca 2+ ] c can also be modulated by calcium influx through voltage-dependent Ca 2+ channels. Many therapies for cardiological diseases are based on the manipulation of voltage-dependent Ca 2+ channels. Thus, cal- cium channel blockers are used by millions of patients to treat hypertension, angina, and heart rhythm abnormalities. These drugs prevent calcium influx into cells and maintain [Ca 2+ ] c at low levels, thereby allowing relaxation of vascular M.V.K. and W.B.B. were each supported by a VA Merit Award and by a gift from the American Legion of Georgia. 1 Current affiliation: Department of Pharmacology, University of Louisiana, New Orleans, Louisiana. 2 Current affiliation: VA Western NY Healthcare System, Buffalo, New York. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. http://dx.doi.org/10.1124/jpet.111.188151. ABBREVIATIONS: [Ca 2+ ] c , cytosolic calcium concentration; ER, endoplasmic reticulum; TRAIL, tumor necrosis factor -related apoptosis- inducing ligand; PBS, phosphate-buffered saline; siRNA, small interfering RNA; CGP-37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-ben- zothiazepin-2(3H)-one; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). 1521-0103/12/3413-646–655 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 341, No. 3 U.S. Government work not protected by U.S. copyright 188151/3769005 JPET 341:646–655, 2012 646 at ASPET Journals on September 26, 2017 jpet.aspetjournals.org Downloaded from