Apoptosis 2004; 9: 437–447 C 2004 Kluwer Academic Publishers
Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics N. Hail, Jr. and R. Lotan Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA
Deguelin exhibits chemopreventive properties in animal carcinogenesis models. The mechanism underpinning the chemopreventive effects of deguelin has not been fully elucidated. However, it has been suggested that this agent reduces ornithine decarboxylase activity, and perhaps the activity of other signaling intermediates associated with tumorigenesis, by inhibiting mitochondrial bioenergetics. We sought to determine if deguelin could trigger apoptosis by inhibiting mitochondrial bioenergetics. Therefore, we compared and contrasted the effects of deguelin on cells from two human cutaneous squamous cell carcinoma cell lines (parental cells) and their respiration-deficient clones lacking mitochondrial DNA (ρ 0 ). While deguelin promoted marked apoptosis in the parental cells in a dose- and time-dependent manner, it failed to do so in the ρ 0 clones. Furthermore, shortterm exposure to deguelin diminished oxygen consumption by the parental cells and promoted mitochondrial permeability transition as evidenced by the dissipation of mitochondrial inner transmembrane potential, reactive oxygen species production, cardiolipin peroxidation, caspase activation, and mitochondrial swelling. Mitochondrial permeability transition was not observed in the ρ 0 clones exposed to deguelin. These results demonstrate that deguelin induces apoptosis in skin cancer cells by inhibiting mitochondrial bioenergetics and provide a novel mechanism for the putative anticancer activity of this agent. Keywords: apoptosis; cancer chemoprevention; deguelin; mitochondria; mitochondrial bioenergetics; mitochondrial permeability transition.
Supported in part by a Cancer Prevention Fellowship (to N. Hail) sponsored by the NCI Grant R25 CA57780; and the Irving and Nadine Mansfield and Robert David Levitt Cancer Research Chair and USPHS Project Grant PO1 CA68233 from the NCI (to R. Lotan).
Correspondence to: N. Hail, Department of Blood and Marrow Transplantation, Box 448, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 770304095, USA. Tel.: (713) 539-7824; Fax: (713) 794-4747; e-mail:
[email protected]
Introduction Deguelin (Figure 1), rotenone, and related compounds are derived from Derris and Lonchorcarpus species and collectively classified as rotenoids.1 These agents constitute the active ingredients in the commercial botanical pesticide cub`e resin.2 The pesticidal action and toxicity of rotenoids result from the inhibition of enzyme-coupled oxidation of NADH, reduction of coenzyme Q, and proton translocation via the mitochondrial NADH:ubiquinone oxidoreductase (EC 1.6.99.3) commonly known as complex I.3 Rotenoids may also prove to be useful in the prevention or treatment of cancer. For example, rotenone has been shown to decrease the incidence of chemically induced tongue carcinoma in rats,4 and spontaneous liver tumor formation in mice.5 Furthermore, deguelin was effective in reducing the incidence of chemically induced skin tumors in mice,6 mammary tumors in rats,6 colonic aberrant crypt foci in mice,7 and preneoplastic lesion formation in mouse mammary gland in organotypic culture.8 The mechanism through which deguelin inhibits carcinogenesis has not been fully elucidated. However, it has been suggested that the suppression of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthesis pathway, may be involved.8 Deguelin has been shown to inhibit 12-Otetradecanoylphorbol-13-acteate (TPA)-induced ODC activity in mouse skin,6 and in cultured cells.1,8–11 Furthermore, deguelin also reduced ODC mRNA steady-state levels in cultured cells.8–10 The decrease of ODC activity by deguelin is contingent on the inhibition of mitochondrial bioenergetics, which is achieved via its pesticidal action in disrupting enzyme-coupled processes in complex I.1,9–11 The modulation of ODC activity by deguelin and other rotenoids in cultured cells11 is not a unique phenomenon given that structurally unrelated inhibitors of complex I can promote similar effects albeit with varying degrees of potency.1,9,10 Apoptosis induction is arguably the most potent defense against cancer. Most chemotherapeutic agents Apoptosis · Vol 9 · No 3 · 2004
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N. Hail, Jr. and R. Lotan Figure 1. Chemical structures of deguelin and rotenone.
Materials and methods Cell culture and reagents
inhibit tumor cell proliferation by inducing apoptosis.12,13 Apoptosis induction is also a desirable attribute of candidate cancer chemopreventive agents,14 including deguelin.15 Two recent studies have reported that deguelin can trigger apoptosis in tumor cells in vitro.15,16 In colon cancer cells, deguelin apparently caused the disregulation of the cell cycle checkpoint protein retinoblastoma (Rb). This activity promoted cell cycle arrest in the colon cancer cells, which was followed by apoptosis induction.15 Deguelin has also been shown to decrease the activity of the phosphatidylinositol 3-kinase/Akt pathway in premalignant and malignant human bronchial epithelial cells, which was believed to trigger apoptosis in these cells.16 In these cell systems, the deguelin-induced suppression of Rb 15 and Akt 16 appeared to be caused by the decreased phosphorylation of these signaling intermediates. This would imply that diminished bioenergetic activity, achieved via the deguelin-mediated inhibition of complex I, was possibly intimately associated with the events leading to apoptosis induction in these cells. Interestingly, several chemopreventive agents 17,18 and chemotherapeutic agents 12,19,20 can target the mitochondria and trigger the downstream effectors of apoptosis directly, irrespective of the upstream control mechanisms (e.g., p53 and pro-apoptotic Bcl-2 family members) or the status of caspases (cysteine proteases cleaving at aspartic acid residues) and/or endogenous caspase inhibitors. Several studies have demonstrated that deguelin inhibits mitochondrial bioenergetics by targeting complex I,1,3,9,10 and other studies have shown that mitochondrial dysfunction can promote apoptosis.17,21–23 Therefore, we examined the hypothesis that deguelin triggers apoptosis by inhibiting mitochondrial bioenergetics. To this end, we compared and contrasted its effects on human cutaneous squamous cell carcinoma (SCC) cells and their respiration-deficient derivatives lacking mitochondrial DNA (ρ 0 clones). Our results highlight apoptosis via the inhibition of mitochondrial bioenergetics as a novel and direct mechanism for deguelin’s anticancer activity. 438 Apoptosis · Vol 9 · No 3 · 2004
The COLO 16 cell line was derived from a metastatic lesion in a female patient who succumbed to metastatic SCC.24 The SRB-12 cell line was derived from cells taken from an epidermal lesion on a patient undergoing skin cancer treatment at the University of Texas M. D. Anderson Cancer Center. The ρ 0 clones of COLO 16 and SRB-12 cells were isolated and characterized as described previously.18,25 Keratinocytes were cultured in Dulbecco’s Modified Eagle’s Medium containing 4.5 mg/ml glucose (Sigma Chemical Co., St. Louis, MO) supplemented with 110 µg/ml pyruvate, 50 µg/ml uridine (both from Sigma Chemical Co.), and 2% fetal bovine serum (Life Technologies, Grand Island, NY). Cell cultures were incubated at 37◦ C in humidified air containing 5% CO2 . Treatment with deguelin or other agents was performed on sub-confluent cultures. Deguelin [(7aS , BaS )-13, 13a-dihydro-9,10dimethoxy-3,3-dimethyl-3H -bis[1] benzopyrano[3,4b:6 ,5 -e ]pyran-7(7aH )-one] was purchased from Microsource Discovery Systems, Inc. (Gaylordsville, CT). A 20 mM stock solution of deguelin in dimethyl sulfoxide (DMSO) was prepared under amber lighting and stored at −70◦ C because deguelin is labile and rapidly decomposes in light and air.8 Working solutions were prepared weekly from this stock solution, which were diluted to the appropriate concentrations in the culture medium for cell treatment. Rotenone, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and DMSO were purchased from Sigma Chemical Co. Dihydroethidium, 10-N-nonyl acridine orange (NAO), and 3,3 -dihexyloxacarbocyanine iodide [DiOC6 (3)] were purchased from Molecular Probes, Inc. (Eugene, OR).
Assays for apoptosis DNA fragmentation was determined using a hypotonic solution of propidium iodide (PI).17 The cells were treated with the specified concentrations of deguelin, rotenone, or an equal volume of the vehicle DMSO for the indicated times, harvested by trypsinization, and combined with their respective culture media that was removed prior to trypsinization. The cells were then pelleted by centrifugation and the cell pellet was resuspended in 1 ml of hypotonic PI solution (50 µg/ml PI, 0.1% sodium citrate, and 0.1% Triton X-100 [all from Sigma Chemical Co.] dissolved in phosphate-buffered saline [PBS] at 4◦ C) and stored overnight at 4◦ C. The cell suspensions were analyzed the next day for PI fluorescence intensity by flow cytometry.
Deguelin induces apoptosis by inhibiting mitochondrial bioenergetics
Concurrent determinations of mitochondrial inner transmembrane potential (m ) dissipation and superoxide production were adapted from a previously published method22 with modifications.17 Briefly, cells in 10cm plastic tissue culture plates were treated with 5 µM deguelin or 10 µM CCCP for the indicated times. Control cultures received an equal volume of the vehicle DMSO. Twenty min before the cells were harvested, DiOC6 (3) and dihydroethidium were added directly to the culture medium to final concentrations of 30 nM and 5 µM, respectively. The cells were harvested by trypsinization, washed with 5 ml PBS at 37◦ C, pelleted by centrifugation, resuspended in 1 ml PBS at 37◦ C, and analyzed immediately for DiOC6 (3) and ethidium fluorescence intensity by flow cytometry. Mitochondrial cardiolipin was measured via the cellular retention of NAO according to a previously published method.26 The cells in 10-cm plastic tissue culture plates were treated for 6 h with 5 µM deguelin or an equal volume of the vehicle DMSO. Thirty min before the cells were harvested, NAO was added directly to the culture medium to final concentration of 100 nM. The cells were harvested as described above and analyzed immediately for NAO fluorescence intensity by flow cytometry. Caspase activity was detected with PhiPhiLuxG1 D2 (Oncoimmunin Inc., Gaithersburg, MD), a cell permeant fluorogenic caspase substrate (DEVD-rhodamine) that is cleaved in a DEVD-dependent manner to produce fluorescent molecules of rhodamine.17,22 The cells in 10-cm plastic tissue culture plates were treated for 6 h with 5 µM deguelin or an equal volume of the vehicle DMSO. The cells were harvested as described above, incubated in 50 µl of 10 µM PhiPhiLux-G1 D2 reagent, and washed with flow cytometry buffer (Oncoimmunin Inc.) according to the manufacturer’s recommendations. The resulting cell suspensions were analyzed immediately for rhodamine fluorescence intensity by flow cytometry.
Measurement of oxygen consumption in cultured cells Oxygen consumption was measured polarographically using a Clark-type oxygen electrode and YSI Model 5300 Biological Oxygen Monitor (Yellow Spring Instrument Co., Yellow Springs, OH) as described previously.17 Briefly, deguelin (5 µM) or an equal volume of the vehicle DMSO was added directly to the medium of cells cultured in 10cm plastic tissue culture plates for a 30-min exposure. The cells were harvested as described above, resuspended at a density of approximately 2 × 106 cells/ml in fresh medium at 37◦ C, and 3 ml of the cell suspension was placed in a 3-ml respiration chamber suspended in a circulating water bath at 37◦ C. Oxygen consumption was measured over a 10-min period after equilibration of the electrode in the respiration chamber. Oxygen consumption rates (nmol O2 /min) were normalized for 10 6 cells assuming an O2 concentration of 220 µM in air-saturated medium at 37◦ C.27
Electron microscopy Cells were seeded in single-well tissue culture slides and allowed to attach and proliferate for 24 to 48 h. The culture medium was removed and replaced with fresh medium containing 5 µM deguelin or an equal volume of the vehicle DMSO. After the indicated times, the medium was removed from the wells and the cells were washed twice with 3 ml of PBS at 37◦ C. The last wash was replaced with 2 ml of fixative (3% glutaraldehyde and 2% paraformaldehyde dissolved in 0.1 M sodium cacodylate [all from Sigma Chemical Co.]; pH 7.3 at 37◦ C). The samples were stored overnight at 4◦ C, embedded, sectioned, and stained using standard electron microscopy procedures. The samples were viewed using a JEOL 1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA) and images were acquired using an Advanced Microscopy Techniques imaging system (Advanced Microscopy Techniques Corp., Danvers, MA).
Flow cytometry All flow cytometric procedures were performed on a Coulter XL flow cytometer and data analysis was accomplished using System II XL software (Coulter Corp., Miami, FL). The experiments employing flow cytometry were typically conducted using duplicate samples for each treatment, unless otherwise indicated, and each experiment was repeated at least three times. Approximately 10,000 cells were evaluated for each sample. In all cytofluorometric determinations, cell debris and cell clumps were excluded from the analysis of the cell suspensions by gating.
Results Deguelin promotes apoptosis in SCC cells Deguelin1,2,8,10,11 and several potent synthetic benzopyran-based inhibitors of complex I28 have been reported to inhibit the proliferation of various types of cancer cells. It has been suggested that the antiproliferative effect of deguelin is associated with its ability to suppress ODC activity in cultured cells.1,2,6,8–11 However, since proliferation inhibition typically required deguelin concentrations in the low micromolar (
