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Mitofusin 1 Degradation Is Induced by a Disruptor of Mitochondrial Calcium Homeostasis, CGP37157: a Role in Apoptosis in Prostate Cancer Cells

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Mitofusin 1 Degradation Is Induced by a Disruptor of Mitochondrial Calcium Homeostasis, CGP37157: a Role in Apoptosis in Prostate Cancer Cells INTERNATIONAL JOURNAL OF ONCOLOGY 44: 1767-1773, 2014 Mitofusin 1 degradation is induced by a disruptor of mitochondrial calcium homeostasis, CGP37157: A role in apoptosis in prostate cancer cells VIVEK CHOUDHARY1-3, ISMAIL KADDOUR-DJEBBAR1-3, RABEI ALAISAMI3, M. VIJAY KUMAR1,2,4 and WENDY B. BOLLAG1,3 1Charlie Norwood VA Medical Center; 2Section of Urology, Department of Surgery, 3Department of Physiology, Medical College of Georgia at Georgia Regents University, Augusta, GA, USA Received January 5, 2014; Accepted February 24, 2014 DOI: 10.3892/ijo.2014.2343 Abstract. Mitochondria constantly divide (mitochondrial fission) apoptotic response of LNCaP cells to CGP, suggesting a likely and fuse (mitochondrial fusion) in a normal cell. Disturbances pro-survival role for Mfn1 in these cells. Our results suggest in the balance between these two physiological processes may that manipulation of mitofusins may provide a novel therapeutic lead to cell dysfunction or to cell death. Induction of cell death advantage in treating prostate cancer. is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of Introduction a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mito- Prostate cancer is the second leading cause of cancer death chondrial calcium efflux, in prostate cancer cells. However, the among men in the United States, with an estimated 30,000 regulation and role of mitochondrial fusion proteins in the death deaths among American men in 2014 (American Cancer of these cells have not been examined. Therefore, our objective Society, www.cancer.org). For effective treatment of pros- was to investigate the effect of CGP on a key mitochondrial tate cancer it is necessary to identify effective and specific fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in pros- molecular targets. As cancer cells exhibit various degrees of tate cancer cell apoptosis. We used various prostate cancer cell mitochondrial dysfunction (1-4), it is of interest to determine lines and western blot analysis, qRT-PCR, siRNA, M30 apop- whether therapeutic strategies focused on mitochondria can be tosis assay and immunoprecipitation techniques to determine developed to preferentially kill cancer cells. mechanisms regulating Mfn1. Treatment of prostate cancer Mitochondria are dynamic organelles that play essential cells with CGP resulted in selective degradation of Mfn1. Mfn1 roles in cellular metabolism, homeostasis and regulation of ubiquitination was detected following immunoprecipitation of cell death. Increasing evidence has demonstrated that changes overexpressed Myc-tagged Mfn1 protein from CGP-treated in mitochondrial morphology are important determinants of cells, and treatment with the proteasomal inhibitor lactacystin, mitochondrial function (1,5). Mitochondria range from long as well as siRNA-mediated knockdown of the E3 ubiquitin filamentous structures to small punctate spheres in different cell ligase March5, protected Mfn1 from CGP-induced degradation. types and under different conditions in the same cell type (6,7). These data indicate the involvement of the ubiquitin-proteasome Mitochondrial structure is affected by the interplay between pathway in CGP-induced degradation of Mfn1. We also demon- the opposing fission and fusion processes that occur normally strated that downregulation of Mfn1 by siRNA enhanced the in a cell (8). Formation of extensive mitochondrial networks (generated by fusion) is considered important for efficient intracellular energy transfer into different cell compartments (9), whereas the punctate mitochondria (arising from fission) seem to assist, in some cases, the induction of apoptosis (10-14). Correspondence to: Dr Wendy B. Bollag, Department of Physiology, Mitochondrial fission involves the large GTPase, Medical College of Georgia at Georgia Regents University, 1120 15th dynamin-related protein 1 (Drp1) and fission 1 (Fis1) (1). Street, Augusta, GA 30912, USA Recent discoveries suggest that mitochondrial fission factor E-mail: [email protected] (Mff), MIEF1/MiD51 and Mid49 act as novel mitochondrial Present address: 4Southern Arizona VA Health Care System, receptors for Drp1 to regulate mitochondrial dynamics in 3601 S 6th Avenue, Tucson, AZ 85723, USA mammalian cells (15-19). Mitochondrial fusion of the outer membranes is controlled by mitofusin 1 (Mfn1) and mito- Key words: mitochondrial fusion, mitofusin 1, ubiquitination, fusin 2 (Mfn2) whereas optic atrophy 1 (OPA1) is implicated prostate cancer, apoptosis in fusion of inner mitochondrial membranes (20,21). Mitofusins are large transmembrane GTPases of the mitochondrial outer membrane, which facilitate the fusion of these membranes in 1768 CHOUDHARY et al: Mfn1 DEGRADATION FOR PROSTATE CANCER CELL DEATH a GTPase-dependent manner (1,22-25). Overexpression of Cells were maintained in RPMI-1640 (HyClone, Logan, UT) Mfn1 results in the formation of characteristic networks of containing 9% FBS (v/v), 0.5% penicillin-streptomycin (v/v), interconnected mitochondria (24), whereas loss of mitofusin and 0.1% fungizone (v/v). The CWR-R1 cells (provided by function causes mitochondrial fragmentation and dysfunction Dr Elizabeth Wilson, University of North Carolina, Chapel (1,24,26-28). In the nervous system, overexpression of Mfn1 can Hill, NC) were grown in Richter's MEM. Non-tumorigenic protect against NO-induced neuronal cell death (29). In yeast, it prostate epithelial cells (P69, a gift from Dr Leland Chung) has been shown that Mdm30p ligase promotes ubiquitination of were maintained in T-medium (Gibco-Invitrogen, Carlsbad, Fzo1p, a yeast homologue of mammalian Mfn1, and its subse- CA). Cells were treated with cycloheximide (50 µg/ml) or CGP quent degradation by the 26S proteasome (30,31). Recently, (10-80 µM) for the indicated period of time. Transfection with March5, an E3 ubiquitin ligase, has been shown to be involved siRNA was performed using HiPer-Fect reagent (Qiagen) for in mammalian Mfn1 degradation in HeLa and Chang cells (32). 48 h followed by treatment with various drugs. Cells were In recent years, agents that impact mitochondria and transiently transfected with the myc-tagged Mfn1 overexpres- exhibit anticancer activity are gaining attention as possible sion plasmid (Myc-Mfn1) by electroporation using the Electro cancer therapies. CGP37157 (CGP) inhibits the efflux of mito- Square Porator™ ECM-830 (BTX Inc., San Diego, CA) as chondrial calcium through the sodium/calcium exchanger, described previously (11). After electroporation, cells were thereby increasing calcium levels in mitochondria and altering seeded in normal growth medium for 24 h and then used in their function. Earlier, we demonstrated that CGP treatment experiments. increases the interaction between Drp1 and Fis1 to induce mitochondrial fission (14), while CGP when combined with Protein extraction and western blot analysis. Appropriately TNF-α related apoptosis-inducing ligand (TRAIL), sensitizes treated cells were washed once with 1X PBS followed by TRAIL-resistant prostate cancer cells to undergo apoptosis addition of lysis buffer [150 mM NaCl, 50 mM Tris-HCl, (33). We have reported that androgen upregulates the expres- pH 8.0, 1 mM EDTA, 0.7% (v/v) Triton X-100, 0.25% (w/v) sion and levels of Drp1, a protein involved in mitochondrial sodium deoxycholate, 1 mM NaF, 1 mM sodium pyrophos- fission, but does not by itself induce mitochondrial fission or phate, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, apoptosis (11). However, treatment of LNCaP cells with CGP in 10 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 10 µg/ml apro- the presence of androgen increases mitochondrial fission and tinin] at 4˚C. Cells were incubated on ice for 30 min and lysates apoptosis (11), suggesting that the androgen-induced increase centrifuged at 10,000 x g at 4˚C for 10 min. The supernatants in Drp1 is not sufficient to trigger mitochondrial fission and were collected, and the protein concentration was estimated apoptosis but requires a second signal to affect mitochondrial using a Bio-Rad DC protein reagent (Bio-Rad Laboratories, function and enhance these processes. Although we demon- Hercules, CA). Proteins (25 µg) were separated on 10 or 12% strated a concomitant decrease in the fusion protein Mfn1, the (w/v) SDS-polyacrylamide gels and transferred to (PVDF) mechanism by which CGP affected Mfn1 protein expression membranes (Bio-Rad Laboratories) using a BioRad semi-dry was not investigated. Therefore, our objective was to investi- transfer apparatus. The blots were blocked in 5% (w/v) non-fat gate the regulation of the mitochondrial fusion protein, Mfn1, dry milk in TBS containing 0.1% (v/v) Tween-20 and incubated in response to CGP treatment as well as its potential role in overnight at 4˚C with primary antibody. Immunoreactive bands prostate cancer cell apoptosis. were visualized using an ECL detection system (Amersham, Pharmacia Biotech, Arlington Heights, IL) on an 8900 Alpha Materials and methods Innotech Image Analyzer (San Leandro, CA) and/or exposed to Hyperfilm and developed. The membranes were re-probed Reagents. The mammalian Myc-tagged Mfn1 overexpression with antibody against β-actin and/or GAPDH, which were plasmid (Myc-Mfn1; a kind gift from Alexander van der Bliek, used as loading controls. Antibodies were diluted in 3% (w/v) University of California, Los Angeles, CA) was used for BSA in TBST. ubiquitination studies. Antibodies used were: anti-Mfn1 (Novus Biologicals,
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