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Volume 270, Number 11, Issue of March 17, 1995 pp. 5830-5838
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protection by Naringin and Some Other Flavonoids of Hepatocytic Autophagy and Endocytosis against Inhibition by Okadaic Acid (*)

(Received for publication, October 5, 1994)

Paul B. Gordon ,  Ingunn Holen ,  Per O. Seglen (§)

From the Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In isolated rat hepatocytes, the protein phosphatase inhibitor okadaic acid exerts a strong inhibitory effect on autophagy, which can be partially overcome by certain protein kinase inhibitors like the isoflavone genistein. To see if other, more specific okadaic acid antagonists could be found among the flavonoids, 55 different flavonoids were tested for their effect on okadaic acid-inhibited autophagy, measured as the sequestration of electroinjected [^3H]raffinose. Naringin (naringenin 7-hesperidoside) and several other flavanone and flavone glycosides (prunin, neoeriocitrin, neohesperidin, apiin, rhoifolin, kaempferol 3-rutinoside) offered virtually complete protection against the autophagy-inhibitory effect of okadaic acid. Unlike genistein, these compounds had little or no autophagy-inhibitory effect of their own. Their innocuousness appeared to be related to glycosylation, because the corresponding aglycones (naringenin, eriodictyol, hesperetin, apigenin, kaempferol) were all inhibitory, in particular apigenin (80% inhibition at 100 µM). Naringin, the most potent okadaic acid-antagonistic flavonoid, gave half-maximal protection at 5 µM and maximal effect at 100 µM. Naringin also prevented the okadaic acid-induced inhibition of endogenous, autophagic lysosomal protein degradation and of receptor-mediated asialoglycoprotein uptake and degradation. Naringin and other okadaic acid-antagonistic flavonoids may be useful tools in the study of intracellular protein phosphorylation and could have potential therapeutic value as protectants against pathological hyperphosphorylations, environmental toxins, or side effects of chemotherapeutic drugs.

INTRODUCTION

Autophagy, the process whereby cells sequester part of their cytoplasm and transport it to lysosomes for degradation, is a basal mechanism used by all cells to obtain amino acids under conditions of amino acid starvation(1, 2) . Because autophagy reduces the cell mass, the growth rate of most cells is inversely correlated with their autophagy rate(3) . The initial sequestration step of the autophagic pathway is subject to regulation by a variety of agents, such as growth factors and hormones, cyclic nucleotides, calcium, and protein phosphorylation(4, 5) . Agents that induce hyperphosphorylation of cellular proteins, like okadaic acid and other protein phosphatase inhibitors, have been shown to suppress autophagy completely in rat hepatocytes, possibly with a causal relation to the disorganization of the cytoskeleton that is also observed with these inhibitors(6, 7) . Both the cytoskeletal and the autophagy-suppressive effects of okadaic acid can be prevented by various protein kinase inhibitors, including KN-62, an inhibitor of Ca/calmodulin-dependent protein kinase II (CaMK-II)(^1)(6) . CaMK-II or related protein kinases would thus seem to be involved in the regulation of autophagy and cytoskeletal organization in hepatocytes.

Several inhibitors of tyrosine-protein kinases (tyrphostin, erbstatin, quercetin) were shown to inhibit autophagy rather than to antagonize the autophagy-inhibitory effect of okadaic acid(6) . One exception was the isoflavone genistein, which antagonized okadaic acid at low concentrations while inhibiting autophagy at higher concentrations(6) . Because genistein can inhibit serine/threonine-protein kinases(8) , histidine-protein kinase(9) , and tyrosine-protein kinases(10) , the possibility was considered that different aspects of flavonoid structure might account for specificity toward different protein kinases involved in positive as well as negative control of autophagy. A large number of flavonoid compounds were therefore investigated in an attempt to find agents capable of antagonizing okadaic acid without inhibiting autophagy. As documented in the present report, several flavonoids were found to exhibit the desired properties, most notably naringin, a glycosidic flavanone.

EXPERIMENTAL PROCEDURES

Cell Preparation and Incubation

Hepatocytes were isolated from 18-h-starved male Wistar rats (250-300 g) by two-step collagenase perfusion(11) . The cells were washed and suspended in suspension buffer (11) containing 15 mM pyruvate and extra Mg (to 2 mM). 2 ml of cell suspension (15-20 mg, cellular wet weight) were incubated at 37 °C in 5-cm albumin-coated plastic Petri dishes.

Autophagy

Autophagic activity was measured as the sequestration of electroinjected [^3H]raffinose (12) in the hepatocytes during incubation at 37 °C. At the end of incubation the cells from each dish were washed twice with 10% sucrose at 0 °C, resuspended in 0.5 ml of 10% sucrose, and electrodisrupted by a single high voltage pulse (2 kV/cm). A 0.3-ml aliquot of the disrupted cell suspension was layered on top of a 4-ml density cushion of buffered metrizamide/sucrose (2.2% sucrose, 8% metrizamide, 50 mM potassium phosphate, 1 mM dithiothreitol, and 1 mM EDTA, pH 7.5). The disrupted cells were sedimented through the cushion by centrifugation for 30 min at 3750 rpm, i.e. approximately 110 times 10^3g times min). Radioactivity in the pellets (autophagocytosed [^3H]raffinose) and disrupted cell samples (total [^3H]raffinose) was measured by liquid scintillation counting. Autophagic activity was expressed as the percentage of [^3H]raffinose sequestered in the pellet relative to the total cellular radioactivity present in the disrupted cell sample.

Uptake and Degradation of I-TC-AOM

For measurements of the uptake and degradation of I-TC-AOM, hepatocytes were incubated for 30 min at 37 °C with okadaic acid (30 nM) before the addition of I-TC-AOM at approximately 50,000 cpm/sample (10 nM); the cells were then incubated further at 37 °C, usually for 2 h. After incubation, the samples were cooled to 0 °C, transferred to 15-ml plastic tubes, and washed once with perfusion buffer (11) containing 10 mM EGTA (final pH 7.5) to remove receptor-bound I-TC-AOM from the cell surface.

After washing, the cell pellets were counted in an LKB -counter to measure the cellular uptake of I-TC-AOM (i.e. the amount of cell-associated radioactivity) expressed as the percentage of the total acid-insoluble radioactivity added initially (because the I-TC-AOM preparation contained 10-20% acid-soluble radioactivity, all cellular radioactivity values are related to acid-insoluble rather than to total initial radioactivity). 1 ml of 10% trichloroacetic acid was then added to each pellet, and the suspension was kept on ice for a minimum of 15 min and thereafter centrifuged for 15 min at 3,700 times g. The supernatant, containing the acid-soluble degradation products of I-TC-AOM, was transferred to a separate tube and counted. The amount of I-TC-AOM degraded was calculated as the percentage of the total acid-insoluble radioactivity added to each sample initially.

Protein Degradation

Protein degradation was measured as the release of acid-soluble radioactivity from protein prelabeled in vivo with [^14C]valine(13) .

Materials

Genistein was purchased from Life Technologies, Inc. (Uxbridge, United Kingdom); galangin, fisetin, and biochanin A were from Aldrich-Chemie GmbH (Steinheim, Germany). Flavone, chrysin, apigenin, baicalein, kaempferol, diosmin, morin, quercetin, quercitrin, rutin, myricetin, naringenin, naringin, hesperetin, hesperidin, rhapontin, malvidin, (+)-catechin,(-)-epicatechin, tetrahydropapaverine, (±)-tetrahydropapaveroline, and DL-laudanosine were from Sigma. Other flavonoids were from Extrasynthese SA (Genay, France). Okadaic acid was purchased from Moana Bioproducts Inc. (Honolulu, HI). KN-62 was obtained from Seikagaku Corp. (Tokyo, Japan), and H-7 was from Sigma. [^3H]Raffinose (5 Ci/mmol, 1 Ci/liter) was from DuPont NEN, and L-[U-^14C]valine (260 Ci/mol, 50 mCi/liter) was from Amersham International (Little Chalfont, United Kingdom).

RESULTS

Protection by Genistein of Hepatocytic Autophagy and Cell Structure against Okadaic Acid

Okadaic acid suppressed hepatocytic autophagy completely even at nanomolar concentrations (Fig. 1), an effect ascribed to inhibition of a type 2A protein phosphatase(7) . The isoflavone genistein inhibited autophagy significantly on its own, as previously observed(6) ; however, it also completely abolished the inhibition by okadaic acid (Fig. 1).

Figure 1: Elimination of the autophagy-inhibitory effect of okadaic acid by genistein. Hepatocytes electroloaded with [^3H]raffinose were incubated at 37 °C for the length of time indicated, with no additions (control, circle), with 15 nM okadaic acid (OA, bullet), with 150 µM genistein (GEN, up triangle), or with both okadaic acid and genistein (GEN + OA, ). After incubation, the cells were electrodisrupted, and the net autophagic accumulation of [^3H]raffinose in sedimentable cell corpses was measured and expressed as the percentage of the total cellular radioactivity. Each value is the mean ± S.E. of three independent experiments.


The effect of okadaic acid on autophagy is paralleled by a disruption of the hepatocytic cytoskeleton, resulting in a structural disintegration of the cell corpses prepared for the autophagy assay (6) . Table 1shows that genistein could offer full protection against this structural effect of okadaic acid, being at least as effective in this respect as KN-62, a specific inhibitor of CaMK-II(14) . H-7, which inhibits protein kinase C potently and the cyclic nucleotide-dependent protein kinases less potently(15) , had no protective effect on hepatocyte structure.

Okadaic Acid-antagonistic and Autophagy-inhibitory Effects of Various Flavonoids

Because the okadaic acid-antagonistic effect of genistein is not shared by some other tyrosine-protein kinase inhibitors like quercetin, tyrphostins, and an erbstatin analog, which rather tend to inhibit autophagy(6) , we surmised that inhibition of some other type of protein kinase might be involved. In an attempt to find inhibitors with better specificity (i.e. with less autophagy-inhibitory effect) than genistein, a systematic screening of other flavonoids from various structural subclasses (Fig. 2) was undertaken. Each flavonoid was initially tested at 100 µM for autophagy-inhibitory and okadaic acid-antagonistic effects; those that exhibited specific antagonism were then subjected to a more detailed dose-response study.

Figure 2: Structures of flavonoids. Upperleft, apigenin (4`,5,7-trihydroxyflavone); upperright, genistein (4`,5,7-trihydroxyisoflavone); lowerleft, naringin (4`,5,7-trihydroxyflavanone 7-hesperidoside); lowerright, naringenin (4`,5,7-trihydroxyflavanone).


The dose-response curves in Fig. 3exemplify some of the reactivity patterns observed. Certain flavonoids were largely inactive (rutin, astragalin) (Fig. 3A); others were predominantly autophagy-inhibitory, with little or no okadaic acid-antagonistic effect (quercitrin, kaempferol) (Fig. 3B). The dose-response curve for genistein was biphasic, with okadaic acid antagonism dominating in the lower dose range and autophagy inhibition in the higher dose range (Fig. 3C). A number of flavonoids were found to be relatively specific okadaic acid antagonists, with no separate effect on autophagy except at the highest concentrations, such as rhoifolin, apiin (Fig. 3D), prunin, kaempferol-3-rutinoside (Fig. 3E), neoeriocitrin, and naringin (Fig. 3F). Naringin showed significant okadaic acid antagonism already at 3 µM and offered complete protection at 100 µM; this compound has, therefore, been accorded particular attention.

Figure 3: Dose-response characteristics of autophagy inhibition and okadaic acid antagonism by various flavonoids. Hepatocytes electroloaded with [^3H]raffinose were incubated for 3 h at 37 °C with flavones at the concentrations indicated in the presence (closedsymbols) or absence (opensymbols) of okadaic acid (15 nM). After incubation, the cells were electrodisrupted, and the net autophagic accumulation of [^3H]raffinose in sedimentable cell corpses was measured and expressed as the percentage of the total cellular radioactivity. A, rutin, single experiment (circle, bullet); astragalin, single experiment (up triangle, ). B, quercitrin, mean ± S.E. of two experiments (circle, bullet); kaempferol, single experiment (up triangle, ). C, genistein, single experiment or mean ± S.E. of two to three experiments (circle, bullet). D, rhoifolin, mean ± S.E. of two to four experiments (circle, bullet); apiin, single experiment at 10, otherwise mean ± S.E. of three experiments (up triangle, ). E, prunin, single experiment (circle, bullet); kaempferol 3-rutinoside, single experiment (up triangle, ). F, neoeriocitrin, single experiment or mean ± S.E. of two experiments (circle, bullet); naringin, mean ± S.E. of two to three experiments (up triangle, ).


Flavones

Table 2summarizes the autophagy-inhibitory and okadaic acid-antagonistic effects of various flavones (2-phenyl-benzopyrones) (Fig. 2). Some of the flavones were strong autophagy inhibitors (flavone, apigenin, 7,3`,4`-trihydroxyflavone, fisetin, luteolin, quercetin); others (chrysin, acacetin, galangin) exhibited nonspecific toxicity (plasma membrane damage) as indicated by a loss of [^3H]raffinose from electroloaded cells. All of the inhibitory and toxic flavones were aglycones, suggesting that conjugation with a sugar may reduce the cytotoxicity of flavonoids. However, not all flavone aglycones were inhibitory (baicalein, 5,7,8-trihydroxyflavone, morin, myricetin), indicating that the position and number of the hydroxyl groups are also important.

Most of the flavones exhibited no significant okadaic acid antagonism, but four were highly antagonistic at concentrations around 250-300 µM: apiin, rhoifolin, kaempferol 7-neohesperidoside, and kaempferol-3-rutinoside (cf. Fig. 3). All of these were hydroxylated at the 5, 7, and 4` positions. A clear okadaic acid-antagonistic effect was also seen with baicalein, but this flavone could not be tested at concentrations higher than 200 µM due to limited solubility. It is noteworthy that three of the effective antagonists were conjugates with disaccharides (apiosylglucose or neohesperidose) containing the unusual 1`-2` bond(16) .

Isoflavones

The effects of isoflavones (3-phenyl-benzopyrones) (Fig. 2) are shown in Table 3. None of the isoflavones tested were particularly autophagy-inhibitory at 100 µM. At higher concentrations some inhibition was observed with genistein, and biochanin A became cytotoxic. Genistein, genistin, biochanin A, and prunetin were significantly okadaic acid-antagonistic at 100 µM, but their effects were not improved at higher concentrations, as illustrated by the increasing predominance of the autophagy-inhibitory effect of genistein (Fig. 3C). An improved antagonism at higher concentrations was indicated for sissotrin, but, because some inhibition of autophagy was also indicated, neither sissotrin nor any of the other isoflavones was subjected to further investigation. All of the okadaic acid-antagonistic isoflavones were hydroxylated at the 5, 7, and 4` positions.

Flavanones

Table 4summarizes the effects of the flavanones (2,3-dihydroflavones) (Fig. 2) tested, all of which were hydroxylated at the 5, 7, and 4` positions. The aglycones (naringenin, isosakuranetin, eriodictyol, hesperetin) tended to inhibit autophagy moderately, although they had little or no okadaic acid-antagonistic effect. The rutinosides (narirutin, eriocitrin, hesperidin) had no effect on autophagy and were inactive or moderately active as okadaic acid antagonists. On the other hand, the 7-glucoside of eriodictyol was strongly okadaic acid-antagonistic at 100 µM, although it had little or no effect on autophagy when given alone at this concentration (at a higher concentration, some inhibition of autophagy was observed). Another glucoside, prunin (naringenin-7-glucoside), had a minimal effect on autophagy on its own but antagonized the inhibition by okadaic acid nearly completely (Fig. 3E).

The most striking and specific effects were observed with the flavanone 7-neohesperidosides (rhamnosylglucosides). Naringin and neoeriocitrin (Fig. 3F) as well as neohesperidin (poncirin apparently being an exception) virtually eliminated the effect of okadaic acid, while affecting autophagy minimally when given alone. Naringin appeared to be particularly potent, exerting its maximal effect at 100 µM and achieving a half-maximal reversal of the okadaic acid inhibition at 5 µM (Fig. 3F).

Miscellaneous Flavonoids

A few structurally complex flavonoids were tested, among which the diflavonoid amentoflavone was found to be a strong inhibitor of autophagy (Table 5). Apart from a moderate autophagy inhibition by rhapontin, none of the other compounds in Table 4exhibited any significant effect. In light of the striking okadaic acid-antagonistic effects of the 7-neohesperidosides, the disaccharide neohesperidose was also tested, but this sugar had no effect in either the absence or the presence of okadaic acid.

Okadaic Acid Dose-dependent Antagonistic Effect of Naringin on Autophagy

Because naringin was the most potent and also one of the most specific okadaic acid antagonists found, it was selected for further investigation. As shown in Fig. 4, the effect of naringin depended on the concentration of okadaic acid used. At 10-15 nM okadaic acid, naringin at 100 µM protected autophagy virtually completely. However, higher concentrations of okadaic acid could overcome the naringin effect, with essentially no antagonism being observed at 200 nM okadaic acid. A higher naringin concentration (1 mM) was only marginally more effective at high okadaic acid concentrations (results not shown).

Figure 4: Okadaic acid dose-dependent antagonistic effect of naringin on hepatocytic autophagy. Hepatocytes electroloaded with [^3H]raffinose were incubated for 3 h at 37 °C with okadaic acid at the concentration indicated in the presence (bullet) or absence (circle) of naringin (100 µM). After incubation, the cells were electrodisrupted, and the net autophagic accumulation of [^3H]raffinose in sedimentable cell corpses was measured and expressed as the percentage of the total cellular radioactivity. Each value is the mean ± S.E. of two experiments (one experiment at 3 times 10M).


Antagonistic Effects of Naringin and Okadaic Acid on Hepatocytic Protein Degradation

Approximately two-thirds of the protein degradation that takes place in amino acid-deprived hepatocytes is due to the autophagic lysosomal pathway(17) . A strong inhibitory effect of okadaic acid on protein degradation would therefore be expected. As shown in Fig. 5, okadaic acid at 15 nM inhibited protein degradation by about 50%. This corresponds to a two-thirds inhibition of the autophagic-lysosomal pathway, recognizable as the fraction of overall protein degradation inhibited by the autophagy inhibitor 3-methyladenine (Fig. 5, dashedline) or by the lysosome inhibitor propylamine (Fig. 5, dottedline). In the presence of these inhibitors, okadaic acid has been shown to have no additional effect (i.e. no effect on non-lysosomal protein degradation)(7) . Naringin completely eliminated the protein degradation-inhibitory effect of okadaic acid (Fig. 5) in accordance with its okadaic acid-antagonistic effect on autophagic sequestration.

Figure 5: Protection of lysosomal protein degradation by naringin against inhibition by okadaic acid. Hepatocytic protein was labeled by an intravenous injection of [^14C]valine (50 µCi) to animals 24 h before isolation of hepatocytes. The cells were incubated for 3 h at 37 °C with (bullet) or without (circle) okadaic acid (15 nM) in the presence of naringin at the concentration indicated. The net release of acid-soluble ^14C radioactivity during the incubation was measured and expressed as the percentage of the initial total cellular radioactivity. The dashed and dottedlines indicate the mean levels of non-autophagic and non-lysosomal protein degradation observed in the presence of 3-methyladenine (10 mM) and propylamine (10 mM), respectively. Each symbol represents the mean ± S.E. of three independent experiments.


Protective Effects of Naringin and KN-62 against Inhibition of Endocytosis by Okadaic Acid

Fig. 6shows that okadaic acid affects endocytosis as well as autophagy. The receptor-mediated endocytic uptake of I-TC-AOM was moderately inhibited (Fig. 6A), and its sedimentability was strongly reduced (Fig. 6B), indicating a reduction in endosomal size or anchorage to the cytoskeleton. Naringin offered nearly complete protection against these effects of okadaic acid (Fig. 6, A and B), although it had little or no effect of its own on endocytosis. The degradation of I-TC-AOM was markedly inhibited by okadaic acid, an effect that could be completely overcome by naringin (Fig. 7).

Figure 6: Naringin antagonism of endocytosis-inhibitory okadaic acid effects. Hepatocytes were incubated with a tracer amount of I-TC-AOM for 3 h (A) or 2 h (B) with (bullet) or without (circle) okadaic acid (20-30 nM) in the presence of naringin at the concentration indicated. The net uptake of I-TC-AOM in whole cells (A) or highly sedimentable cell corpses (B) was measured and expressed as the percentage of the total amount of radioactivity added to the incubate. Each value is the mean of triplicate samples from a single experiment.


Figure 7: Effects of okadaic acid, naringin, and KN-62 on degradation of endocytosed I-TC-AOM. A, hepatocytes were incubated at 37 °C for the length of time indicated with a tracer amount of I-TC-AOM in the presence (filledsymbols) or absence (opensymbols) of okadaic acid (30 nM) and with no further additions (circle, bullet), with 10 µM KN-62 (up triangle, ), or with 100 µM naringin (box, ). After incubation, the cells were washed and precipitated with 10% trichloroacetic acid, and the acid-soluble I radioactivity was measured and expressed as the percentage of the total cell-associated radioactivity. Each value is the mean ± S.E. of two to three experiments. B, hepatocytes were incubated for 3 h at 37 °C with a tracer amount of I-TC-AOM in the presence (filledsymbols) or absence (opensymbols) of okadaic acid (30 nM) with naringin (circle, bullet) or KN-62 (up triangle, ) at the concentration indicated. The net amount of acid-soluble I radioactivity released was measured and expressed as the percentage of the total cell-associated radioactivity. Each value is the mean of triplicate samples from a single experiment.


The okadaic acid-antagonistic effects of flavonoids on hepatocytic autophagy and cytoskeletal organization are shared by KN-62, a specific inhibitor of CaMK-II(6) . However, KN-62 did not offer a protection of endocytosis equivalent to that provided by naringin (Fig. 7). At high concentrations, KN-62 had an inhibitory effect of its own on I-TC-AOM degradation that outweighed the okadaic acid-antagonistic effect seen at lower concentrations (Fig. 7B). KN-62 inhibited endocytic protein degradation without the lag seen with okadaic acid (Fig. 7A), possibly indicating a more direct effect on endocytosis.

DISCUSSION

Flavonoids occur ubiquitously in the plant kingdom and are common components of the human diet(18) . The flavonoids exhibit a wide structural diversity; more than 4,000 different flavonoids have been identified from various plants. Flavonoids have been shown to have structurally dependent, highly specific effects on a variety of enzymes and are able to interfere with numerous cellular processes, including growth and differentiation (see (19) for review). The diversity of flavonoid effects may relate to their structural similarity to ATP and hence to their ability to compete with ATP for binding to various enzymatic sites(20) .

Like most flavonoids, naringin has metal-chelating, antioxidant, and free radical scavenging properties (21, 22, 23) and may offer some protection against mutagenesis (24) and lipid peroxidation(25) . The ability of naringin to inhibit certain isoforms of cytochrome P-450 may account for its effects on procarcinogen activation and drug metabolism (26) . Naringin has been reported to suppress the development of inflammatory lung edema in experimental animals (27) and to inhibit the growth of certain fungi(28) . However, in most biological systems studied, naringin has little or no effect on parameters sensitive to other flavonoids, such as glucose transport(29) , chloride transport (30) , leukocytic secretion(31) , prostaglandin synthesis(32) , protein kinase C activity(33) , Ca-ATPase activity(31) , or the growth of various cell types in culture(34, 35) . The high potency and effectiveness of naringin as an okadaic acid antagonist observed in the present study is therefore relatively unique.

Because the only mechanism of action known for okadaic acid is as an inhibitor of protein phosphatases, its biological effects can be ascribed to the hyperphosphorylation of cellular proteins, shown to be extensive in isolated hepatocytes(36) . Naringin and the other active flavonoids are therefore most likely to antagonize okadaic acid by functioning as protein kinase inhibitors, in analogy with the strong antagonism shown by certain well established kinase inhibitors like K-252a and KN-62(6) . The metal-chelating and antioxidant properties of naringin would seem unlikely to be involved, because these would be shared by non-antagonists like quercetin, kaempferol, fisetin, etc. Although no protein kinase-inhibitory effect of naringin has yet been demonstrated directly, other flavonoids have been shown to inhibit a variety of protein kinases, e.g. receptor tyrosine-protein kinases like the epidermal growth factor receptor and the platelet-derived growth factor receptor, and soluble tyrosine-protein kinases like the Src protein(10, 20, 37) . Furthermore, flavonoids have been found to inhibit histidine-protein kinase (9) and several serine/threonine-protein kinases, like S6-kinase(8) , myosin light chain kinase(38, 39) , protein kinase C(33, 38) , and casein kinase II (40) . The cyclic AMP-dependent protein kinase holoenzyme is apparently not affected by flavonoids(10, 38) , but its catalytic subunit is strongly inhibited by a large number of these compounds(41) . Many flavonoids may thus have the potential for competitive inhibition at the ATP-binding site of protein kinases, but their access, and thus their specificity, is apparently restricted by the three-dimensional structure of the intact holoenzymes.

The effective okadaic acid antagonism displayed by KN-62, a reportedly specific inhibitor of CaMK-II(14) , would seem to implicate the latter enzyme as mediating the okadaic acid-induced inhibition of autophagy. However, the difference between the actions of naringin and KN-62 on hepatocytic endocytosis may indicate that the putative naringin-sensitive kinase is actually a different, perhaps closely related, enzyme. The parallel effects of okadaic acid and its antagonists on autophagy and cytoskeletal structure (6) suggest that the alterations in autophagic activity may be secondary to changes in the phosphorylation of cytoskeletal elements. CaMK-II as well as many other protein kinases appear to be involved in cytoskeletal protein phosphorylation(42, 43, 44) ; there is, therefore, no lack of candidate enzymes.

Among the structural features of the flavonoid molecule required for okadaic acid antagonism, glycosylation seemed to be particularly important, probably by preventing inhibition of autophagy, which would otherwise mask the antagonism (except in the cases of the isoflavones, which had little inhibitory ability to begin with). Glycosylation in the 7 position appeared to be most effective, but some 3-glycosides (kaempferol 3-rutinoside and quercitrin) were also antagonistic. The nature of the sugar was important, contributing to antagonistic effectiveness in the tentative order of neohesperidose, apiosylglucose, glucose, and rutinose (cf. the apigenin, naringenin, and eriodictyol series). The role of the hydroxylation pattern was more difficult to assess, because all the flavone and flavanone glycosides available were hydroxylated in both the 5 and 4` positions. Among the isoflavones, replacement of the 5-OH group with a 6- or 8-OH group abolished antagonistic activity, whereas some activity was retained in the absence of 5-OH. The 4`-OH apparently needed to be free (cf. the low antagonistic activity of the 4`-methoxylated neohesperidosides fortunellin and poncirin) or supplemented with a 3`-OH group (neohesperidin), except in the isoflavone series.

Flavonoid inhibition of autophagy was most evident with the flavone aglycones, the isoflavones being largely inactive at 100 µM and the flavanone aglyones having only low or moderate inhibitory effects. Although the non-hydroxylated parent compound flavone was a strong inhibitor, a free 4`-OH group seemed to be essential for inhibitory activity among the hydroxylated flavonoids (cf. the inactivity of compounds in which the 4`-OH group was blocked (acacetin) or absent (galangin, baicalein)). On the other hand, 2` or 5` hydroxylation (morin, myricetin) seemed to reduce the autophagy-inhibitory ability, whereas 3` hydroxylation (fisetin, luteolin) did not. Hydroxylation in the 4` position was also found to be essential for flavonoid inhibition of promutagen-activating liver enzymes(45) , for DNA binding and topoisomerase II-catalyzed DNA damage (46) , and, most interestingly, for inhibition of epidermal growth factor receptor kinase(47) . Inhibition of a tyrosine-protein kinase could be a plausible mechanism for the suppression of hepatocytic autophagy by flavonoids, because several non-flavonoid tyrosine-protein kinase inhibitors also inhibit autophagy(6) .

Both autophagy-inhibitory flavonoids, like apigenin, fisetin, luteolin, and amentoflavone, and okadaic acid antagonists, like naringin, prunin, neoeriocitrin, neohesperidin, apiin, rhoifolin, and kaempferol-3-rutinoside, may be useful in experimental studies of autophagy and other okadaic acid-sensitive cellular processes. In addition, the okadaic acid-antagonistic flavonoids could have potential value as protectants against hyperphosphorylating environmental toxins (48, 49) , pathological hyperphosphorylations(50, 51) , or the side effects of cytotoxic drugs used in the clinic.

FOOTNOTES

*
This project has been generously supported by the Norwegian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: CaMK-II, Ca/calmodulin-dependent protein kinase II; I-TC-AOM, I-tyramine cellobiose-asialoorosomucoid.

ACKNOWLEDGEMENTS

We thank Mona Birkeland for providing excellent technical assistance. I-TC-AOM was kindly given to us by Prof. Trond Berg.

REFERENCES

  1. Mortimore, G. E. (1987) in Lysosomes: Their Role in Protein Breakdown (Glaumann, H., and Ballard, F. J., eds) pp. 415-443, Academic Press, London
  2. Seglen, P. O., and Bohley, P. (1992) Experientia (Basel) 48, 158-172 [CrossRef][Medline] [Order article via Infotrieve]
  3. Ballard, F. J. (1987) in Lysosomes: Their Role in Protein Breakdown (Glaumann, H., and Ballard, F. J., eds) pp. 285-318, Academic Press, London
  4. Seglen, P. O., Gordon, P. B., Holen, I., Høyvik, H., Kov á cs, A. L., Strømhaug, P. E., and Berg, T. O. (1992) in Endocytosis (Courtoy, P. J., ed) pp. 247-254, Springer-Verlag, Berlin
  5. Seglen, P. O., Holen, I., and Gordon, P. B. (1993) in Molecular Mechanisms of Membrane Traffic (Morr é , D. J., Howell, K. E., and Bergeron, J. J. M., eds) pp. 339-349, Springer-Verlag, Berlin
  6. Holen, I., Gordon, P. B., and Seglen, P. O. (1992) Biochem. J. 284, 633-636
  7. Holen, I., Gordon, P. B., and Seglen, P. O. (1993) Eur. J. Biochem. 215, 113-122 [Medline] [Order article via Infotrieve]
  8. Linassier, C., Pierre, M., Le Pecq, J.-B., and Pierre, J. (1990) Biochem. Pharmacol. 39, 187-193 [CrossRef][Medline] [Order article via Infotrieve]
  9. Huang, J., Nasr, M., Kim, Y., and Matthews, H. R. (1992) J. Biol. Chem. 267, 15511-15515 [Abstract/Free Full Text]
  10. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 5592-5595 [Abstract/Free Full Text]
  11. Seglen, P. O. (1976) Methods Cell Biol. 13, 29-83 [Medline] [Order article via Infotrieve]
  12. Seglen, P. O., Gordon, P. B., Tolleshaug, H., and Høyvik, H. (1986) Exp. Cell Res. 162, 273-277 [CrossRef][Medline] [Order article via Infotrieve]
  13. Seglen, P. O., Grinde, B., and Solheim, A. E. (1979) Eur. J. Biochem. 95, 215-225 [Medline] [Order article via Infotrieve]
  14. Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M., and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320 [Abstract/Free Full Text]
  15. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041 [CrossRef][Medline] [Order article via Infotrieve]
  16. Horowitz, R. M., and Gentili, B. (1963) Tetrahedron 19, 773-782 [CrossRef]
  17. Seglen, P. O. (1983) Methods Enzymol. 96, 737-764 [Medline] [Order article via Infotrieve]
  18. Hertog, M. G. L., Hollman, P. C. H., Katan, M. B., and Kromhout, D. (1993) Nutr. Cancer 20, 21-29 [Medline] [Order article via Infotrieve]
  19. Brandi, M. L. (1992) Bone and Miner. 19, S3-S14
  20. Graziani, Y., Erikson, E., and Erikson, R. L. (1983) Eur. J. Biochem. 135, 583-589 [Medline] [Order article via Infotrieve]
  21. Jung, G., Hennings, G., Pfeifer, M., and Bessler, W. G. (1983) Mol. Pharmacol. 23, 698-702 [Abstract]
  22. Kroyer, G. (1986) Z. Ernaehrwiss. 25, 63-69 [CrossRef][Medline] [Order article via Infotrieve]
  23. Chen, Y. T., Zheng, R. L., Jia, Z. J., and Ju, Y. (1990) Free Radical Biol. & Med. 9, 19-21
  24. Francis, A. R., Shetty, T. K., and Bhattacharya, R. K. (1989) Carcinogenesis 10, 1953-1955 [Abstract/Free Full Text]
  25. Maridonneau-Parini, I., Braquet, P., and Garay, R. P. (1986) Pharmacol. Res. Commun. 18, 61-72
  26. Guengerich, F. P., and Kim, D. H. (1990) Carcinogenesis 11, 2275-2279 [Abstract/Free Full Text]
  27. Lambev, I., Krushkov, I., Zheliazkov, D., and Nikolov, N. (1980) Eksp. Med. Morfol. 19, 207-212 [Medline] [Order article via Infotrieve]
  28. Zu, Y.-L., Wu, F., Gilchrist, A., Ai, Y., Labadia, M. E., and Huang, C.-K. (1994) Biochem. Biophys. Res. Commun. 200, 1118-1124 [CrossRef][Medline] [Order article via Infotrieve]
  29. Kimmich, G. A., and Randles, J. (1978) Membr. Biochem. 1, 221-237 [Medline] [Order article via Infotrieve]
  30. Nguyen, T. D., and Canada, A. T. (1993) J. Nutr. 123, 259-268
  31. Bennett, J. P., Gomperts, B. D., and Wollenweber, E. (1981) Arzneim.-forsch. 31, 433-437 [Medline] [Order article via Infotrieve]
  32. Baumann, J., Wurm, G., and von Bruchhausen, F. (1980) Arch. Pharm. 313, 330-337 [CrossRef]
  33. Ferriola, P. C., Cody, V., and Middleton, E., Jr. (1989) Biochem. Pharmacol. 38, 1617-1624 [CrossRef][Medline] [Order article via Infotrieve]
  34. Bracke, M., Vyncke, B., Opdenakker, G., Foidart, J.-M., De Pestel, G., and Mareel, M. (1991) Clin. & Exp. Metastasis 9, 13-25
  35. Namgoong, S. Y., Son, K. H., Chang, H. W., Kang, S. S., and Kim, H. P. (1994) Life Sci. 54, 313-320 [CrossRef][Medline] [Order article via Infotrieve]
  36. Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81 [CrossRef][Medline] [Order article via Infotrieve]
  37. Abler, A., Smith, J. A., Randazzo, P. A., Rothenberg, P. L., and Jarett, L. (1992) J. Biol. Chem. 267, 3946-3951 [Abstract/Free Full Text]
  38. Rogers, J. C., and Williams, D. L., Jr. (1989) Biochem. Biophys. Res. Commun. 164, 419-425 [CrossRef][Medline] [Order article via Infotrieve]
  39. Jinsart, W., Ternai, B., and Polya, G. M. (1991) Hoppe-Seyler's Z. Physiol. Chem. 372, 819-827
  40. Cochet, C., Feige, J. J., Pirollet, F., Keramidas, M., and Chambaz, E. M. (1982) Biochem. Pharmacol. 31, 1357-1361 [CrossRef][Medline] [Order article via Infotrieve]
  41. Jinsart, W., Ternai, B., and Polya, G. M. (1992) Hoppe-Seyler's Z. Physiol. Chem. 373, 205-211
  42. Vallano, M. L., Goldenring, J. R., Lasher, R. S., and DeLorenzo, R. J. (1986) Ann. N. Y. Acad. Sci. 466, 357-374 [Abstract]
  43. Baumann, K., Mandelkow, E.-M., Biernat, J., Piwnica-Worms, H., and Mandelkow, E. (1993) FEBS Lett. 336, 417-424 [CrossRef][Medline] [Order article via Infotrieve]
  44. Blackshear, P. J. (1993) J. Biol. Chem. 268, 1501-1504 [Free Full Text]
  45. Lee, H., Wang, H.-W., Su, H.-Y., and Hao, N. J. (1994) Mutagenesis 9, 101-106 [Abstract/Free Full Text]
  46. Austin, C. A., Patel, S., Ono, K., Nakane, H., and Fisher, L. M. (1992) Biochem. J. 282, 883-889
  47. Ogawara, H., Akiyama, T., Watanabe, S., Ito, N., Kobori, M., and Seoda, Y. (1989) J. Antibiotics 42, 340-343 [Medline] [Order article via Infotrieve]
  48. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. (1990) Trends Biochem. Sci. 15, 98-102 [CrossRef][Medline] [Order article via Infotrieve]
  49. Carmichael, W. W. (1994) Sci. Am. 270, 78-86 [Medline] [Order article via Infotrieve]
  50. Grundke-Iqbal, I., Iqbal, K., Tung, Y.-C., Quinlan, M., Wisniewski, H. M., and Binder, L. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4913-4917 [Abstract/Free Full Text]
  51. Liu, J., Mathias, S., Yang, Z., and Kolesnick, R. N. (1994) J. Biol. Chem. 269, 3047-3052 [Abstract/Free Full Text]
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