(Received for publication, October 5, 1994)
From the
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
[H]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.
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)(
)(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.
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
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.
Figure 1:
Elimination of the autophagy-inhibitory
effect of okadaic acid by genistein. Hepatocytes electroloaded with
[H]raffinose were incubated at 37 °C for the
length of time indicated, with no additions (control,
),
with 15 nM okadaic acid (OA,
), with 150
µM genistein (GEN,
), or with both okadaic
acid and genistein (GEN + OA,
). After incubation,
the cells were electrodisrupted, and the net autophagic accumulation of
[
H]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.
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 [H]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 [
H]raffinose in
sedimentable cell corpses was measured and expressed as the percentage
of the total cellular radioactivity. A, rutin, single
experiment (
,
); astragalin, single experiment (
,
). B, quercitrin, mean ± S.E. of two experiments
(
,
); kaempferol, single experiment (
,
). C, genistein, single experiment or mean ± S.E. of two
to three experiments (
,
). D, rhoifolin, mean
± S.E. of two to four experiments (
,
); apiin, single
experiment at 10
, otherwise mean ± S.E. of
three experiments (
,
). E, prunin, single
experiment (
,
); kaempferol 3-rutinoside, single experiment
(
,
). F, neoeriocitrin, single experiment or mean
± S.E. of two experiments (
,
); naringin, mean
± S.E. of two to three experiments (
,
).
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) .
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).
Figure 4:
Okadaic acid dose-dependent antagonistic
effect of naringin on hepatocytic autophagy. Hepatocytes electroloaded
with [H]raffinose were incubated for 3 h at 37
°C with okadaic acid at the concentration indicated in the presence
(
) or absence (
) of naringin (100 µM). After
incubation, the cells were electrodisrupted, and the net autophagic
accumulation of [
H]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
10
M).
Figure 5:
Protection of lysosomal protein
degradation by naringin against inhibition by okadaic acid. Hepatocytic
protein was labeled by an intravenous injection of
[C]valine (50 µCi) to animals 24 h before
isolation of hepatocytes. The cells were incubated for 3 h at 37 °C
with (
) or without (
) okadaic acid (15 nM) in the
presence of naringin at the concentration indicated. The net release of
acid-soluble
C 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.
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 (
) or without (
) 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 (
,
), with 10 µM KN-62 (
,
), or with 100
µM naringin (
,
). 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 (
,
) or KN-62
(
,
) 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.
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.