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* Department of Biology, University of Maryland, College Park, Maryland; and Department of Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas
Correspondence: Address reprint requests to Marco Colombini, Tel.: 301-405-6925; Fax: 301-314-9358; E-mail: colombini{at}umd.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jayatilake et al., 2003). For example, the avicins have been shown to induce apoptosis by direct perturbation of mitochondria (Haridas et al., 2001b; Mujoo et al., 2001), activating the intrinsic caspase pathway. Recent results (V. Haridas, X. X. Li, M. Higuchi, M. Colombini, and J. U. Gutterman, unpublished data) demonstrate that the avicins favor the closure of the outer mitochondrial membrane channel, VDAC, resulting in suppression of nucleotide exchange and decrease in oxygen consumption. Postmitochondrial events during apoptosis include suppression of HSP70 and HSP90 by activation of the ubiquitin/proteasome pathway (A. Gaikwad, A. Poblenz, V. Haridas, and J. U. Gutterman, unpublished data). Avicins' proapoptotic effects are accompanied by a cytoprotective effect on healthy cells. The NRF-2 transcriptional factor is activated by a redox mechanism and thus stimulates expression of a gene battery enhancing cell detoxification and antioxidant effects (Haridas et al., 2004). As part of the regulation of stress, the avicins also suppress multiple proinflammatory components of the innate immune system, including the transcriptional factor NF-
B (Haridas et al., 2001a), the Pi3K/AKT signaling pathway, as well as heat shock proteins. In mouse skin models, the avicins have been shown to suppress the initiation and promotion phase of chemical carcinogenesis as well as UVB damage with resultant suppression of oxidative DNA and lipid damage (Hanausek et al., 2001). Thus, the avicins represent a new class of metabolites that regulate cellular redox balance through a coordinated system response to cellular stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and stored refrigerated in aliquots appropriate for one day of use. Dry aliquots were generated by dissolving avicin in water and lyophilizing. Working solutions (1 mg/ml) to be used on the same day were generated by freshly dissolving the avicin in the appropriate medium, generally the same medium used on the cis side of the planar membrane experiment (see below). The solution was kept on ice until needed. All other reagents were purchased from either Fisher Scientific (Pittsburgh, PA) or Sigma Chemical (St. Louis, MO).
Reconstitution experiments in planar phospholipid membranes
Planar phospholipid membranes were formed by the monolayer method of Montal and Mueller (1972) as revised by Colombini (1987). A phospholipid membrane was formed across a 0.1-mm-diameter hole in a Saran partition using monolayers composed of phospholipids (either DPhyPC or asolectin, or a combination of these, plus or minus cholesterol as indicated). The membrane separated two 5-mL aqueous compartments named cis and trans containing 0.10 M KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.0 (unless otherwise indicated). The voltage was applied to the cis side, and the trans side was maintained at virtual ground by an amplifier in the inverted mode (52 K; Analog Devices, Norwood, MA). The current was recorded both on a chart recorder and with the Axotape recording system (Vers. 2, Axon Instruments, Foster City, CA). The current was filtered at a frequency of 20 Hz using a Butterworth filter. Calomel electrodes with built-in saturated KCl bridges were used to interface with the aqueous solutions.
Erythrocyte lysis assay
Erythrocytes were obtained from decapitated rats (Sprague-Dawley males) with added EGTA to prevent clotting. The cells were washed by successive centrifugations from 150 mM NaCl, 4 mM EGTA, and 5 mM HEPES, pH 7.4 and resuspended in the same buffer. The erythrocyte suspension was diluted 1:20 with an equiosmolal solution of the test non-electrolyte. The final erythrocyte concentration was such that 400 µL of it would result in an absorbance of 0.15 at 540 nm after reaction with the Drabkins reagent (Sigma Technical Bulletin No. 525, Sigma Chemical). To 500 µL of the erythrocyte suspension, 5 µL of avicin (1 mg/mL) was added and the cells sedimented (2 min at 12,000 g) after a prescribed period of time (generally 5 min). A 400-µL aliquot of supernatant was mixed with 400 µL of Drabkins reagent and the absorbance at 540 nm measured after 5 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Channel formation may be the result of the presence of the acyclic monoterpine side chain. The addition of a modified version of avicin G in which the side chain was cleaved (Fig. 1) resulted in no detectable formation of channels in the membrane up to a final concentration in the aqueous phase of 25 µg/ml (five times the amount needed to see a conductance with avicin G). Beyond that, an increase in membrane conductance was observed but it had very different properties. A smooth increase in conductance characteristic of a carrier was observed (data not shown). Thus, rather than the formation of channel-like pathways, the truncated molecule may have resulted in the transport of ions, one or a few ions at a time.
The increase in conductance with addition of normal avicin G was obviously nonlinear (Fig. 3). Further additions increased the conductance in a logarithmic fashion, indicating that multiple avicin G molecules were required to generate the permeability pathway. From the slopes of the logarithmic plots that ranged from 4 to 12, the entire process is highly cooperative.
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Size of the aqueous pathway formed by avicin G channels
The size of these channels was determined by adding avicin G to erythrocytes. The cells were suspended in iso-osmotic solutions consisting of non-electrolytes of different molecular size. The percent lysis was measured by sedimenting the intact cells and measuring hemoglobin released from lysed cells. If the avicin channels are permeable to the test non-electrolyte, the resulting osmotic imbalance will lyse the erythrocytes. Fig. 6 shows that the cutoff was between polyethylene glycol (PEG) 1000 and PEG 1500. In the inset, one can see that, with time, more lysis was observed with PEG 1000, but that lysis in the presence of PEG 1500 leveled off, indicating a slow permeation by PEG 1000. Doubling the dose of avicin did not change the size cutoff. The Stokes-Einstein radii and the radii of gyration of these molecules are 1.0 and 1.2 nm for PEG 1000 and PEG 1500, respectively. The clear cutoff shows that avicin G forms channels of a discrete size.
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To quantitatively estimate the relative potency of the avicins in different lipid environments, we calculated the dose required to obtain a conductance of 10 nS. This was calculated for each separate experiment and averaged (Fig. 10). Note that only avicin G in asolectin showed a very strong cholesterol dependence on its ability to form channels. Other trends were not statistically significant. These differences in potency are also evident in Fig. 7. Note that if a higher conductance was used as a benchmark, the preference for channel formation in asolectin over DPhyPC would be much more evident.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The avicins seem to be in the latter category. A key to understanding their mode of action may lie in their ability to form channels in specific membranes in cells. In this work we have sought to characterize the channels formed by avicins and to begin exploring the conditions that favor or disfavor channel formation. Avicins are water-soluble, but are sufficiently amphipathic to partition into membranes. Unlike detergents, avicins increase the membrane permeability to ions without destabilizing the membranes to any significant extent. The observed conductance depends on the amount of avicin added to the aqueous phase resulting in clear, stable conductance levels. This behavior is characteristic of amphipathic structures that self-assemble in the membrane to form well-defined structural entities.
Erythrocytes provide a convenient system to measure the pore size of the avicin channels by using non-electrolytes. Both avicin G and avicin D self-assemble into channels with an estimated pore size of 1.1 nm in radius. Slow erythrocyte lysis in PEG 1000 shows that this molecule's hydrodynamic radius closely matches the narrowest part of the aqueous pore. This was seen with both avicins, indicating a similar channel-forming structure. The fact that the selectivity of avicin channels in membranes containing negatively-charged lipids remained rather constant as the conductance was changed over three orders of magnitude, shows that avicin channels exist at an optimum size, and that increases in conductance reflect increases in the number of channels rather than in the size of channels.
The major difference between avicin G and D is the sensitivity to the presence of cholesterol. The presence of a single additional hydroxyl group in the extended side chain of avicin D replaces the need for cholesterol to increase the propensity for channel formation. Since, without cholesterol, avicin D forms channels of almost identical properties to those formed by avicin G with cholesterol, this indicates that, for avicin G, cholesterol is more of a regulator than an essential part of the channel-forming pathway. Saponins, in general, have been shown to bind to cholesterol (Shany et al., 1974; Osbourn et al, 1996), and thus avicin D may still bind cholesterol—but if it does, the cholesterol is not influencing the propensity to form channels.
The dependence of avicin G channels on cholesterol may explain the ability of avicin G to induce apoptosis selectively in tumor cells. Low levels induce apoptosis in Jurkat cells (Haridas et al., 2001b) but are cytoprotective in normal cells (Haridas et al., 2001a, 2004; Hanausek et al., 2001). The reason for this differential effect may lie in the higher cholesterol content in the mitochondrial outer membranes of tumor cells (Galeotti et al., 1986). The cholesterol content in tumor cell mitochondria is approximately four times that of normal cell mitochondria (Parlo and Coleman, 1984).
Clearly, other differences in the composition of said membranes may be responsible for the observed specificity. In addition, lack of knowledge about the activity of cholesterol in biological membranes (separate from the cholesterol composition) hampers one's ability to predict which membranes might be more sensitive to permeabilization by avicin G. Another factor may be the presence of substances, in solution or on the membrane, that sequester avicin monomers preventing the free avicin from achieving the critical concentration required for channel formation. Nevertheless, the correlation between avicin's channel-forming ability and its ability to induce apoptosis exists, and is strengthened by the observation that truncated avicin G does not induce apoptosis in Jurkat cells (Jayatilake et al., 2003).
Although avicin G can induce cytochrome-c release from mitochondria (Haridas et al., 2001b), our measurements demonstrate that its pore size is too small to allow cytochrome c release. A variety of both anti- and pro-apoptotic regulators form channels so avicin may act in a similar way. Alternatively, avicin may interact with other proteins and lipids that have been implicated as forming the pathways from the selective release of proteins from mitochondria in the early phases of apoptosis: e.g., BAX and ceramide (Schlesinger et al., 1997; Siskind et al., 2002).
From an examination of the structure of the avicins one might not suspect that these would form channels in membranes. The highly glycosylated triterpene nucleus should ensure that it is anchored to the aqueous phase on the surface of the membrane. Thus, the amphipathic extended side chain is an attractive candidate for the transmembrane portion of the avicin that might form the walls of the channel. Indeed, triterpenoid saponins with an acyl side chain have been shown to increase membrane permeability of endothelial cells, whereas those lacking the side chain did not (Melzig et al., 2001). This finding is consistent with our results in that the removal of the acyl side chain of avicin G results in the loss of all channel-like behavior. Thus, we propose that the avicin channels are essentially barrels composed of extended side chains that span the membrane (Fig. 11). The number of these chains would be given by the power dependence of the conductance on the avicin concentration (6–10 depending on the conditions used). On one end of the channel (on the side of avicin addition) the triterpene portions of the molecules must pack together to form some sort of ring structure. This structure may or may not form the end of the permeability pathway. At the other end, headgroups of phospholipids should form an annulus through which permeating ions must pass to cross the membrane. This annulus could be responsible for the selectivity of avicin in negatively-charged asolectin membranes. A related triterpenoid saponin, avenacin A-1, was shown to permeabilize planar phospholipid membranes (Armah et al., 1999), and this conductance was interpreted as being due to channel formation.
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). Note that here also the cells do not undergo necrosis. The details of how this process works are under investigation.
Avicins induce apoptosis and this can be partly explained by a direct effect of avicin favoring VDAC closure (V. Haridas, X. X. Li, M. Higuchi, M. Colombini, and J. U. Gutterman, unpublished data), because closure of VDAC channels has been shown to be an early and reversible event leading to mitochondria-mediated apoptosis (Vander Heiden et al., 2001). It is not obvious, however, how channel formation by avicins can lead to the induction of apoptosis. The following is a speculative proposal that could guide future research. Consider that many Bcl-2 family proteins, both pro- and anti-apoptotic, form channels (Minn et al., 1997; Schlesinger et al., 1997), and the role of channel formation is unclear save for the obvious formation of a pathway for protein flux. The anti-apoptotic protein Bcl-xL forms cation selective channels (Minn et al., 1997), whereas the pro-apoptotic proteins form channels that are either nonselective or weakly anion selective (Schlesinger et al., 1997). Respectable theoretical calculations show that metabolic flux across the mitochondrial outer membrane coupled with differential permeability to the permeating ions (small ions and metabolic ions) can lead to a substantial potential across the mitochondrial outer membrane (Lemeshko and Lemeshko, 2000; Lemeshko, 2002). The potential would act in at least two ways: 1), favor the closure of the voltage-gated VDAC channels located in this membrane; and 2), result in a pH in the intermembrane space that differs from that of the cytosol (protons will equilibrate with the potential). These effects could lead to the initiation of apoptosis by either VDAC closure and/or a pH-dependent activation or inhibition of enzymes leading to apoptosis. An example of the latter could be ceramide-induced apoptosis where the steady-state level of mitochondrial ceramide would be controlled by the relative activities of enzymes that generate and consume ceramide (Siskind et al., 2002). Thus, by changing the selectivity of the mitochondrial outer membrane, avicin could alter the potential across this membrane, leading to conditions favoring apoptosis. Obviously, the complexity of this proposal, and particularly the influence of membrane lipids, allow plenty of room for tumor cells to be hypersensitive to avicin.
Submitted on July 13, 2004; accepted for publication January 5, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Colombini, M. 1987. Characterization of channels isolated from plant mitochondria. Methods Enzymol. 148:465–475.[Medline]
Galeotti, T., S. Borrello, G. Minotti, and L. Masotti. 1986. Membrane alterations in cancer cells: the role of oxy radicals. Ann. N. Y. Acad. Sci. 488:468–480.[CrossRef][Medline]
Hanausek, M., P. Ganesh, Z. Walaszek, C. J. Arntzen, T. J. Slaga, and J. U. Gutterman. 2001. Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model. Proc. Natl. Acad. Sci. USA. 98:11551–11556.
Haridas, V., C. J. Arntzen, and J. U. Gutterman. 2001a. Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), inhibit activation of nuclear factor-B by inhibiting both its nuclear localization and ability to bind DNA. Proc. Natl. Acad. Sci. USA. 98:11557–11562.
Haridas, V., M. Higuchi, G. S. Jayatilake, D. Bailey, K. Mujoo, M. E. Blake, C. J. Arntzen, and J. U. Gutterman. 2001b. Avicins: triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation. Proc. Natl. Acad. Sci. USA. 98:5821–5826.
Haridas, V., M. Hanausek, G. Nishimura, H. Soehnge, W. Zbigniew, M. Narog, E. Spears, R. Zoltaszek, and J. U. Gutterman. 2004. Triterpenoid electrophiles (avicins) activate innate stress response by redox regulation of a gene battery. J. Clin. Invest. 113:65–73.[CrossRef][Medline]
Jayatilake, G. S., D. R. Freeberg, Z. Liu, S. L. Richheimer, M. E. Blake, D. T. Bailey, V. Haridas, and J. U. Gutterman. 2003. Isolation and structures of avicins D and G: potent tumor-inhibiting saponins derived from Acacia victoriae. J. Nat. Prod. 66:779–783.[Medline]
Joshi, L., J. M. Van Eck, K. Mayo, R. DiSilvestro, M. E. Blake, T. Ganapathi, V. Haridas, J. U. Gutterman, and C. J. Arntzen. 2002. Metabolomics of plant saponins: bioprospecting triterpene glycoside diversity with respect to mammalian cell targets. OMICS. 61:235–246.
Lemeshko, S. V., and V. V. Lemeshko. 2000. Metabolically derived potential on the outer membrane of mitochondria: a computational model. Biophys. J. 79:2785–2800.
Lemeshko, V. V. 2002. Model of the outer membrane potential generation by the inner membrane of mitochondria. Biophys. J. 82:684–692.
Massari, P., C. A. King, A. Y. Ho, and L. M. Wetzler. 2003. Neisserial PorB is translocated to the mitochondria of HeLa cells infected with Neisseria meningitidis and protects cells from apoptosis. Cell. Microbiol. 5:99–109.[CrossRef][Medline]
Melzig, M. F., G. Bader, and R. Loose. 2001. Investigations of the mechanism of membrane activity of selected triterpenoid saponins. Plant Med. 67:43–48.[CrossRef]
Minn, A. J., P. Vélez, S. L. Schendel, H. Liang, S. W. Muchmore, S. W. Fesik, M. Fill, and C. B. Thompson. 1997. Bcl-xL forms an ion channel in synthetic lipid membranes. Nature. 385:353–357.[CrossRef][Medline]
Montal, M., and P. Mueller. 1972. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. USA. 69:3561–3566.
Mujoo, K., V. Haridas, J. J. Hoffmann, G. A. Wächter, L. K. Hutter, Y. Lu, M. E. Blake, G. S. Jayatilake, D. Bailey, G. B. Mills, and J. U. Gutterman. 2001. Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis. Cancer Res. 61:5486–5490.
Osbourn, A. E., P. Bowyer, and M. J. Daniels. 1996. Saponin detoxification by plant pathogenic fungi. Adv. Exp. Med. Biol. 404:547–555.[Medline]
Parlo, R. A., and P. S. Coleman. 1984. Enhanced rate of citrate export from cholesterol-rich hepatoma mitochondria. The truncated Krebs cycle and other metabolic ramifications of mitochondrial membrane cholesterol. J. Biol. Chem. 259:9997–10003.
Schlesinger, P. H., A. Gross, X. M. Yin, K. Yamamoto, M. Saito, G. Waksman, and S. J. Korsmeyer. 1997. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic Bcl-2. Proc. Natl. Acad. Sci. USA. 94:11357–11362.
Shany, S., A. W. Bernheimer, P. S. Grushoff, and K. S. Kim. 1974. Evidence for membrane cholesterol as the common binding site for cereolysin, streptolysin O and saponin. Mol. Cell. Biochem. 3:179–186.[CrossRef][Medline]
Siskind, L. J., R. N. Kolesnick, and M. Colombini. 2002. Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins. J. Biol. Chem. 277:26796–26803.
Vander Heiden, M. G., X. X. Li, E. Gottleib, R. B. Hill, C. B. Thompson, and M. Colombini. 2001. Bcl-xL promotes the open configuration of VDAC and metabolite passage through the mitochondrial outer membrane. J. Biol. Chem. 276:19414–19419.
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