ArticlePDF AvailableLiterature Review

Plant Phenols and Autophagy

April 2016
Biochemistry (Moscow) 81(4):297-314

DOI:10.1134/S0006297916040015

Authors:

Anton Chechushkov

Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences

Peter M Kozhin

Research Center of Clinical and Experimental Medicine, Siberian Division of the Russian Academy of Medical Sciences, Russia, Novosibirsk.

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Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular, neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redox-sensitive signaling system and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are presented.

Schematic representation of autophagy: a) initiation; b) elongation; c) autophagosome formation; d) degradation of phagophore conn tent. PE, phosphatidylethanolamine; Ub, ubiquitin (explanations of all other abbreviations are presented in the list of abbreviations and dess ignations).

…

Redox transformations of hydroxytyrosol.

…

Figures - uploaded by Elena Menshchikova

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Content uploaded by Elena Menshchikova

Content may be subject to copyright.

Under normal conditions, reactive oxygen species

(ROS) of radical (О2

•–, NO•, RO•, and others) and non

radical (H2O2, ONOOH, ROOH) nature constitute an

important part of cell regulators, but very often they

become toxic and destructive, especially during patholog

ical processes, and, as a result, oxidative stress develops

[1]. A multilevel system of antioxidants of enzymatic

(superoxide dismutase, catalase, glutathione peroxidase,

etc.) and nonenzymatic (glutathione and other SHcon

taining compounds, phenols, ascorbate) nature exists in

cells to maintain a low level of ROS. For a long time, the

antioxidant effect of phenols was considered from the

point of view of their antiradical action and their ability to

act as chelators of metals of variable valency, thus inter

rupting oxidation reactions occurring via the free radical

chain mechanism [2]. Recently, mechanisms of indirect

protective effect of phenols were identified and thorough

ly studied, in particular their ability to activate the system

of antioxidantresponsive element, Keap1/Nrf2/ARE,

and induce autophagy [35]. Such effect of phenolic

compounds is important for protection against oxidative

and carbonyl stress [69], motivating us to analyze possi

ble mechanisms of the effect of natural phenols on the

Keap1/Nrf2/ARE signaling system and autophagy.

ISSN 00062979, Biochemistry (Moscow), 2016, Vol. 81, No. 4, pp. 297314. © Pleiades Publishing, Ltd., 2016.

Original Russian Text © N. K. Zenkov, A. V. Chechushkov, P. M. Kozhin, N. V. Kandalintseva, G. G. Martinovich, E. B. Menshchikova, 2016, published in Biokhimiya, 2016,

Vol. 81, No. 4, pp. 429447.

REVIEW

297

Abbreviations: Akt, protein kinase B (or PKB); AMPK, AMPactivated protein kinase; ARE, antioxidant respons(iv)e element;

Atg, AuTophaGyrelated genes; BAX, Bcl2associated X protein; Bcl2, Bcell lymphoma 2; BNIP3, Bcl2/adenovirus E1B nine

teen kDa interacting protein 3; Cu,ZnSOD, copper and zinccontaining superoxide dismutase; eIF4EBP1, eukaryotic initiation

factor 4E binding protein 1; ER, endoplasmic reticulum; ERK1/2, extracellular signalregulated protein kinase 1/2; FOXO1, fork

head box protein O1; GABARAP, gammaaminobutyric acid receptorassociated protein; HIF1α, hypoxiainducible factor 1α;

HMGB1, high mobility group box 1; IRE1, inositol requiring kinase 1; JAK, Janus kinase; JNK, cJun Nterminal kinases; Keap1,

Kelchlike ECHassociating protein 1; LC3, microtubuleassociated protein 1A/1Blight chain 3; LDL, lowdensity lipoproteins;

MAPK, mitogenactivated protein kinases; MnSOD, manganesedependent superoxide dismutase; mTOR, mammalian target of

rapamycin (serine/threonine protein kinase); mTORC1/2, mammalian target of rapamycin complex 1/2; Nrf2, NFE2related fac

tor 2; p62/SQSTM1, ubiquitinbinding protein p62, or sequestosome 1; p70S6K, p70S6 kinase (serine/threonine protein kinase);

PERK, protein kinaselike endoplasmic reticulum kinase; PI3K, phosphatidylinositol 3kinase; Ras, rat sarcoma protein; ROS,

reactive oxygen species; SIRT1, sirtuin 1; TSC2, tuberous sclerosis complex 2; ULK1/ULK2, Unc51like kinase 1/2 (serine/thre

onine protein kinase); Vps34, vacuolar protein sortingassociated protein 34 (PI3K class III); Wnt, wingless/integration signaling

pathway.

* To whom correspondence should be addressed.

Plant Phenols and Autophagy

N. K. Zenkov1, A. V. Chechushkov1, P. M. Kozhin1, N. V. Kandalintseva2,

G. G. Martinovich3, and E. B. Menshchikova1*

1Research Institute of Experimental and Clinical Medicine, Novosibirsk 630117,

Russia; fax: +7 (383) 3336456; Email: lemen@centercem.ru

2Novosibirsk State Pedagogical University, 630126 Novosibirsk, Russia; fax: +7 (383) 2441161; Email: nspu@nspu.net

3Belarusian State University, 220030 Minsk, Belarus; fax: +7 (375) 172095267; Email: martinovichgg@bsu.by

Received October 23, 2015

Revision received November 19, 2015

Abstract—Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells

during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular,

neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant

effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redoxsensitive signaling system

and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are pre

sented.

DOI: 10.1134/S0006297916040015

Key words: phenolic antioxidants, autophagy, Keap1/Nrf2/ARE signaling system, regulation

298 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

Keap1/Nrf2/ARE SYSTEM

More than 20 redoxsensitive transcription factors are

now recognized, with the Nrf2 (nuclear E2related factor 2

that regulates expression of genes containing ARE (antiox

idant respons(iv)e element, 5′A/GTGAC/TnnnGCA/G3′)

in the promoters) occupying a special place among them.

Nrf2 in cells is under constant control of the Keap1

(Kelchlike ECH associating protein 1) repressor protein,

which in turn is a sort of molecular “sensor” of intracel

lular redox balance. Intimate association of these molec

ular structures allows combining them into a unified

redoxsensitive system Keap1/Nrf2/ARE with the main

function of maintaining internal homeostasis under

apoptosisinducing, carcinogenic, and stresscausing

conditions. ARE controls expression of more than 500

genes. Two major groups of antioxidant enzymes can be

identified among them: the first group includes heme

oxygenase 1, glutathione peroxidase 2, glutamate cysteine

ligase, glutathione reductase, thioredoxin reductase, and

others, and the second one – enzymes of the second

phase of xenobiotic detoxification (glutathione Strans

ferases A, M, and P, NAD(P)H:quinone oxidoreductase

1, NRH:quinone oxidoreductase 2, UDPglucuronosyl

transferases A and B, and others) [10, 11]. The biological

importance of the Keap1/Nrf2/ARE system that regu

lates intracellular redox balance lies in the fact that it

controls activity of a number of redoxsensitive transcrip

tion factors as well as metabolic processes involving phos

phatases and kinases.

AUTOPHAGY

The term “autophagy” (from Greek “αυ

’το

′ς” – self,

and “ϕαγει

~ν” – to eat) was introduced in 1963 by the

Nobel Prizewinning Belgian cytologist and biochemist

Christian de Duve to describe the process of production

of nutrients as a result of lysosomal catabolism of intra

cellular compartments [12]. Several types of autophagy

have been identified: macroautophagy (formation of a

phagophore with double isolation membrane sequester

ing intracellular structures for fusion with lysosomes),

microautophagy (engulfment of cytoplasmic content by

lysosomal membrane invagination), and chaperonemedi

ated autophagy (damaged molecules are delivered to lyso

somes by chaperone proteins). Macroautophagy is the

main type of autophagy, which is a fairly selective process:

it can be directed at the degradation of protein aggregates

(aggrephagy), damaged mitochondria (mitophagy), per

oxisomes (pexophagy), secretory granules (crinophagy),

lipid vacuoles (lipophagy), as well as intracellular

pathogens, such as bacteria and viruses (xenophagy) [13].

Autophagy is now considered as an important protection

factor during oxidative and carbonyl stress because dam

aged mitochondria and peroxisomes, which are major

sources of ROS in cells, are removed with its help as well

as protein and lipid aggregates whose formation is the a

main sign of cytotoxicity of carbonyl compounds [7, 14].

Moreover, induction of autophagy activates the

Keap1/Nrf2/ARE signaling system and enhances antiox

idative cell protection [15, 16].

The intensity of autophagy depends on availability

and strength of inducers, which include either internal

factors (deficit of nutrients, presence of damaged

organelles, denatured proteins and their aggregates,

oxidative and toxic stress) or external factors such as bac

teria or viruses, interferon γ, and vitamin D3. The induc

tion of autophagy occurs within 1 h following exposure to

strong stimuli; however, the process slows after 24 h. The

serine/threonine protein kinase mTOR (mammalian tar

get of rapamycin) with molecular mass 289 kDa consist

ing of 2549 a.a. is the main intracellular tool to switch off

autophagy [17]. In cells mTOR operates in the composi

tion of the protein complexes mTORC1 (mammalian tar

get of rapamycin complex 1) and mTORC2. mTORC1

controls autophagy, and at the same time is itself under

control of a number of kinase signaling cascades, prima

rily AMPK/ERK/TSC2/mTOR (inhibits mTORC1 and

activates autophagy) and JAK/PI3K/Akt/mTOR (main

tains mTORC1 activity and inhibits autophagy). As a

result, a balance between anabolic and catabolic process

es is achieved. More than 30 proteins are involved in the

activation of autophagy, and its overall picture is extreme

ly complicated and not fully understood. Studies of

Saccharomyces cerevisiae cells revealed 35 genes required

for autophagy, which were combined into a group referred

to as Atg (autophagyrelated genes). Furthermore, many

analogs of the Atg yeast proteins have been found in

mammals.

The autophagy process can be broken down into

stages of initiation, elongation ending with formation of

the autophagosome with isolating double membrane, fus

ing of phagophore with lysosome resulting in formation of

autophagolysosome, and degradation of the phagophore

content (Fig. 1). Initiation of autophagosome formation

is achieved by the ULK1/2 complex with its activity neg

atively controlled by mTORC1 and stimulated by AMPK

[18]. This complex is tailanchored into the ER mem

brane where it in turn activates a kinase complex consist

ing of Beclin 1 (Atg6 in yeast) and Vps34 kinase [19] and

is required for recruiting other proteins for forming the

autophagosome [20, 21]. Among them are proteins

Atg12, Atg5, and Atg16L1, which form a complex that

inhibits ubiquitinlike proteins LC3 (isoforms A, B, and

C are present in mammalian cells) and GABARAP with

phosphatidylethanolamine [22]. Both proteins are

required for membrane elongation of the forming

autophagosome as well as for sequestration of cytoplas

mic material degraded in autophagosomes [23].

Cytoplasmic material (protein aggregates, damaged

organelles, and even pathogens), which has been first

PLANT PHENOLS AND AUTOPHAGY 299

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

marked with one or several receptor types (p62/

SQSTM1, NIX/BNIP3L, etc.), is subjected to packing

into autophagosomes. Following completion of the pack

ing of degraded material, the autophagosome membrane

closes and further can fuse with late endosomes and lyso

somes [18]. Disruption at any stage of autophagy can lead

to fatal consequences for either developing embryos or

mature organisms. Many neurodegenerative diseases are

associated with mutations in the autophagy genes partic

ipating in its realization at different stages [24].

Curcumin (Fig. 2) – the principle curcuminoid in

Curcuma longa L. roots is a yellow pigment that is some

times called Indian saffron or yellow ginger; it has been

used since ancient times in Indian and Southeast Asian

Fig. 1. Schematic representation of autophagy: a) initiation; b) elongation; c) autophagosome formation; d) degradation of phagophore con

tent. PE, phosphatidylethanolamine; Ub, ubiquitin (explanations of all other abbreviations are presented in the list of abbreviations and des

ignations).

a b

d c

complex

ULK1/2

complex

Beclin 1Vps/34

lysosome

protein aggregate damaged organelle pathogen

LC3II (LC3/PE complex) lysosomal enzymes

300 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

medicine, as well as a foodgrade coloring (E100). The

curcuminoid composition of C. longa roots includes in

addition to curcumin (80%) also demethoxycurcumin

(17%) and bisdemethoxycurcumin (3%). Curcumin

exhibits a wide range of biological effects: it activates or

inhibits more than 30 transcription factors, multiple

kinases, and other enzymes, and demonstrates pro

nounced antiinflammatory, immunomodulating, and

anticarcinogenic effects [25]. Moreover, curcumin is

nontoxic even at large doses (12 g per day). Curcumin is a

rather potent antioxidant and is capable of inhibiting

superoxide anion radical (О2

•–), hydroxyl radical (OH•),

singlet oxygen (1O2), hydrogen peroxide (H2O2), nitric

oxide (NO•), peroxynitrite (ONOOH), and peroxyl rad

icals (RO•, RO2

•) [26].

Many biological effects of curcumin are related to its

ability to activate the system of antioxidantresponsive

element Keap1/Nrf2/ARE and to induce the expression

of antioxidant genes [27]. The electrophilic group of cur

cumin with α,βunsaturated carbonyl bonds, which

interacts with cysteine residues of Keap1 as a Michael

acceptor, is essential for activation of the Keap1/Nrf2/

ARE system [28]. This indicates that the phenol OH

groups are not important for the activation effect and

removal of hydroxyl and methoxyl groups from the aro

matic structure do not significantly affect the functions of

curcumin analogs. At the same time, nordihydroguaiaret

ic acid (Fig. 2) that does not have conjugated unsaturated

bonds, but has catechol phenol structure, is an effective

activator of Nrf2 [29]. Nordihydroguaiaretic acid is found

in significant quantities (up to 7%) in the leaves of the

desert plant Larrea divaricata (creosote bush). Through

the use of two catechol structures, nordihydroguaiaretic

acid is able to inhibit either radical (О2

•–, OH•, NO•) or

nonradical (1O2, H2O2, ONOOH) ROS species [30].

Curcumin induces autophagy very efficiently.

Analysis of the effect of curcumin analogs B19 and

MHMD revealed that the phenol OH groups are also not

essential for induction of autophagy [31, 32], which is

confirmed by the lack of induction effect by nordihy

droguaiaretic acid. On the other hand, tetrahydrocur

cumin (a major metabolite of curcumin in cells) [33] and

bisdemethoxycurcumin [34] are effective stimulators of

autophagy, hence the role of conjugated double bonds

and carbonyl groups remains unclear. Mechanisms of ini

tiation of autophagy by curcumin are quite diverse

(table). In a number of publications, the ability of cur

cumin to activate intracellular generation of ROS was

reported; antioxidant Nacetylcysteine decreased and

hydrogen peroxide increased autophagy in the process

[35, 36]. It is suggested that curcumin depolarizes the

mitochondrial membrane, which triggers generation of

ROS and initiation of apoptosis. The role of curcumin in

this process is twofold: on one hand, it stimulates genera

tion of ROS, and on the other hand it scavenges them,

being an antioxidant [26, 37, 38]. Curcumin can activate

or inhibit multiple transcription factors including those

(NFκB, Nrf2, AP1, HIF1) that directly participate in

induction and progress of autophagy [9, 39]. It was shown

in both in vitro and in vivo studies that curcumin

enhanced expression of Beclin 1, which is an inducer of

autophagy [40].

Curcumin is now available in the form of tablets and

capsules and introduced into the composition of drinks,

creams, and various cosmetic products [103]. The major

problem of medicinal application of curcumin is its low

bioavailability: curcumin is unstable at physiological pH 7

and is absorbed poorly in the gastrointestinal tract. It was

possible to increase absorption 30fold by creating phyto

somes (complex of curcumin with phosphatidylcholine

from soya beans) [104]. The Meriva preparation (Italy)

was developed on the basis of phytosomes. Meriva

decreased pain and level of inflammation markers in the

blood serum of patients with chronic osteoarthritis [105].

A fourweek course of Meriva (1 g per day) reduced

swelling in diabetic patients and increased microcircula

tion [106]. Large scale clinical trials were begun and fin

ished in recent years (more than 60 according to the web

site of the National Institutes of Health, USA) that stud

ied tolerance, pharmacokinetics, prophylactic, and treat

ment efficiency of various formulations of curcumin

(purified preparation, liposomal and microgranular

forms, watersoluble form CR011L, as well as in combi

nation with extracts of Ginkgo Biloba and green tea). The

development and investigation of curcumin complexes

with cyclodextrins and nanoparticles that allow improv

ing its stability and solubility are underway.

Stilbenes attracted attention of researchers during

investigation of the “French paradox”, in particular of

relatively low mortality from cardiovascular diseases with

moderate consumption of red grape wines. Multiple stud

ies of this phenomenon resulted in the conclusion that

one of major acting ingredients of grape wines is a

polyphenolic phytoalexin – resveratrol – found in grape

skin and seeds (Fig. 2) [107]. In addition to grapes and

red wine, resveratrol has been found in grains, berries,

nuts, and many other plantderived products. In plants,

stilbenes perform an important protective role: they func

tion as antibacterial and antifungal agents as well as pro

tect from the effects of ultraviolet radiation and ozone as

antioxidants [108].

In animal experiments, resveratrol showed cardio

and neuroprotective effects as well as exhibited strong

protective function in streptozotocininduced type I dia

betes [107, 109]. Despite the fact that resveratrol demon

strates a wide range of biological effects (inhibits tran

scription factors NFκB, AP1, p53, activates kinases

MAPK, Akt, AMPK, PI3K, as well as SIRT1 [107, 108]),

many of its functions are related to the ability to scavenge

radicals and activate the Keap1/Nrf2/ARE signaling sys

tem [108, 110]. For direct antioxidant action, the 4′OH

group is important [111]. The mechanism of resveratrol

PLANT PHENOLS AND AUTOPHAGY 301

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

Fig. 2. Structures of plant phenols.

Curcumin

(1,7bis(4hydroxy3methoxyphenyl)

1,6heptadiene3,5dione)

Nordihydroguaiaretic acid

(4,4’(2,3dimethylbutan

1,4diyl)dibenzene1,2diol)

Resveratrol

(trans3,5,4’trihydroxystilbene) Pterostilbene (4(trans2

(3,5dimethoxyphenyl)ethylene)phenol)

Quercetin

(3,3’,4’,5,7pentahydroxyflavone) Genistein

(4’,5,7trihydroxiisoflavone)

Baicalein

(5,6,7trihydroxyflavone) Fisetin

(5,3’,4’,7tetrahydroxyflavone) (–)Epigallocatechin3gallate

([2R,3R)5,7dihydroxy2

(3,4,5trihydroxyphenyl)chroman3yl]

3,4,5trihydroxybenzoate)

Gallic acid

(3,4,5trihydroxybenzoic

acid)

paraCoumaric acid

(3(4hydroxyphenyl)

2propenoic acid)

Ferulic acid

(3(4hydroxy

3methoxyphenyl)prop2enoic acid)

302 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

Compound,

concentration

Curcumin

1040 μM

540 μM

50 and 100 μM

1050 μM

2 μM

1090 μM

540 μM

1100 μM

140 μM

150 μM

180 μM

30 μM

Resveratrol

25100 μM

100 μM

10 μM

1050 μM

50200 μM

12.5100 μM

1050 μM

200 μM

Refe

rences

[36]

[40]

[41]

[42]

[43]

[44]

[45]

[37]

[35]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

Effect of plant phenols on autophagy

Effect on

autophagy

induction

activation

inhibition

induction

Cell lines

HCT116

A549

ACCMESO1

glioblastoma cells

U87MG, U373MG

K562

A375, G361

YD10B

HUVEC

H9c2 under ischemia/

reperfusion

MCF7

HeLa, 293T

HCT116, HeLa

HUVEC

A549

K562

Molt4, Jurkat, CEM

C115, CEMC714

Mechanism of action

activation of ROS production, kinases ERK1/2 and p38

MAPK do not participate

increase of Beclin 1 and p62 concentration

enhancement of AMPK phosphorylation

enhancement of LC3 expression and induction

of autophagosome formation

inhibition of PI3K/Akt/mTOR signaling pathway

inhibition of Akt/mTOR signaling pathway; activation

of ERK1/2

decreased Bcl2 expression; increase of Beclin 1

and LC3II levels; apoptosis induction

depolarization of mitochondria membranes and enhance

ment of ROS production

activation of ROS production, which is inhibited by N

acetylcysteine

enhancement of FOXO1 acetylation and its binding with

Atg7; inhibition of P13K/Akt/mTOR signaling pathway;

induction of Beclin 1 dissociation from Bcl2; decrease of

H2O2induced apoptosis

induction of Bcl2 expression and inhibition of Bax

expression; increase of expression of Beclin 1, BNIP3,

and SIRT1; suppression of apoptosis

enhancement of Beclin 1 expression; induction of apop

tosis

inhibition of mTOR via activation of SIRT1 or AMPK

activation of SIRT1

increase of cAMP level; activation of AMPK and SIRT1

activation of AMPK/SIRT1 signaling pathway; protection

from toxic effects of oxidized LDL

induction of Ca2+ ions release; activation of AMPK and

inhibition of mTOR

increase of LC3B II formation

enhancement of AMPKdependent mTOR inhibition;

activation of JNK and p62 expression

inhibition of Akt/mTOR phosphorylation and activation

of p38 MAPK

PLANT PHENOLS AND AUTOPHAGY 303

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

250 μM

60300 mg/kg

per day; 25 μM

30 and 100 μM

25100 μM

1040 μM

25150 μM

25100 μM

12.5100 μM

20500 μM

1664 μM

1080 μM

Pterostilbene

1040 μM

1100 μM

50100 μM

10100 μM

1550 μM

0.51 μM

γγTocotrienol

1040 μM

40 μM

0.15 μM

1030 μM

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

Table (Contd.)

activation of p38 MAPK; decrease of H2O2induced

apoptosis

increase of FOXO1 transcriptional activity via activation

of SIRT1

activation of class III PI3K

increase of STIM1 expression and induction of ER stress

inhibition of Wnt/bcatenin signaling pathway

increase of intracellular ROS production

enhancement of ceramide production and inhibition

of Akt/mTOR signaling pathway

decrease of p70 S6 kinase activity

activation of p38 and ERK1/2 kinases, induction does not

depend on PI3K

inhibition of Akt/PKB/mTOR signaling pathway inde

pendent on Beclin 1

activation of cAMPPRKAAMPKSIRT1 signaling

pathway

inhibition of Akt, p38, ERK1/2 kinases and activation

of JNK1/2

decrease of ROS production; increase of autophagosome

accumulation

inhibition of phosphorylation of PI3K/AKT and JNK

kinases; activation of ERK

inhibition of mTOR

increase of H2O2production; induction of intracellular

sterol accumulation

increase of intracellular Ca2+ content, activation of

AMPKα1 and inhibition of mTOR

inhibition of PI3K/Akt/mTOR signaling pathway

induction ER stress and release of Ca2+ ions; increase

of Beclin 1 content

increase of Beclin 1 content and decrease of p62, but not

depend on AMPK

enhancement of synthesis and accumulation of sphin

golipids in cells

induction

inhibition1

induction

disruption

induction

H9c2

U87, U251, U138

PC3, DU145

SUM159, MCF7

HT29, COLO 201

B16

HEK293, HeLa,

U2OS, HEK293T,

NIH/3T3

U373

MCF7

HepG2

SAS, OECM1

HL60

A549

T24

MCF7

HUVEC

MCF7, MDAMD231

hASCs

PC3, LNCaP

304 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

Epigallocatechin

3gallate

10360 μM

220 μM

50500 μM

15240 μM

20 μM

1040 μM

Quercetin

10160 μM

1050 μM

3090 μM

12.5100 μM

Genistein

100 μM

40 μM

50 and 100 μM

Baicalein

12.5100 μM

20, 40 μM

25200 μM

Fisetin

40120 μM

520 μM

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

Table (Contd.)

activation of Ca2+ ion release from ER; activation of

CaMKK and AMPK

induction of HMGB1 (amphoterin) aggregation

suppression of NO•production

enhancement of ROS production; disruption of lysosome

membrane permeability

decrease of ROS production and acetylation of FOXO1

enhancement of AMPK phosphorylation and increase of

its activity, activation of fusion with lysosomes

inhibition of Akt/mTOR and activation of HIF1α

process depends on expression of Ras oncogenic protein

inhibition of proteasomes, increase of protein aggregate

content; inhibition of mTOR/eIF4EBP1; activation of

p70S6K

increase of Beclin 1 expression; inhibition of S6K1 kinase

phosphorylation

decrease of MnSOD, Cu,ZnSOD, and thioredoxin

content; increase of BAX/Bcl2 protein ratio

decrease of Bcl2 content; increase of Beclin 1 content;

induction of apoptosis

inhibition of Akt/mTOR

inhibition of Akt

inhibition of Akt/mTOR

inhibition of mTOR/Raptor; activation of AMPK

induction of ER stress and release of Ca2+ ions

inhibition of mTORC1 activity and decrease of Raptor

and Rictor content and of Akt activity

inhibition of Akt/mTOR signaling pathway

induction

inhibition

inhibition2

induction

induction3

induction

BAEC

RAW264.7

HepG2, MEF

H9c2

Huh7, HepG2

AGS, MKN28

СасоН2

MCF7, MDAMB453,

HeLa, OVCAR3, IM9

HeLa

MCF7

MIA PaCa2

HT29

A2780, CaOV3, ES2

HepG2

PC3, DU145, MDA

MB231

SMMC7721, Bel7402

PC3, DU145, LNCaP

A549

PLANT PHENOLS AND AUTOPHAGY 305

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

effect in activation of the Keap1/Nrf2/ARE signaling sys

tem remains unclear: it either activates Nrf2 expression

and inhibits Keap1 expression, or activates kinases

AMPK, Akt B, and ERK1/2, or affects nuclear transcrip

tion factors [108110, 112]. Investigation of a series of

resveratrol analogs showed that the 3,4dihydroxystilbene

with catechol structure demonstrated the highest Nrf2

inducing activity [111]. It is interesting to note that very

little attention was paid to the dimeric form of resvera

trol – εviniferin – during investigation of the “French

paradox”, while its content in some wine varieties is high

er than that of the resveratrol, and which exhibits higher

Nrf2inducing activity probably due to activation of ERK

and p38 MAPK kinases [113].

The effect of resveratrol on autophagy can be real

ized via multiple mechanisms similar to curcumin (table).

Furthermore, the resveratrol effect depends on the type of

cell culture and used concentrations; resveratrol usually

exhibits cytotoxic effect at concentrations above 100 μM

[114]. The ability of resveratrol to activate sirtuin 1

(SIRT1) [4951], which induces autophagy by deacety

lating Atg proteins (Atg5, Atg7, Atg8), transcription fac

tors FOXO1, FOXO3, p53, NFκB, and activating

AMPK [115, 116] was shown in multiple studies. The in

vitro studies show that twofold enhancement of the

deacetylating activity of SIRT1 is observed at 11 μM con

centration of resveratrol, and saturation is achieved at

100200 μM of resveratrol [117]. Resveratrol is not a spe

cific sirtuin activator, many plant phenols demonstrate

such ability; moreover, a direct relationship is observed

between deacetylation of cellular proteins and activation

of autophagy [115, 118]. Activation of kinases (AMPK,

JNK, p38 MAPK) by the increase of intracellular con

centrations of Ca2+ and cAMP is an important mecha

nism of resveratrol action [51, 53, 108]. At the same time,

resveratrol can change activity of kinases as a result of

60 μM

Gallic acid

150 μM

paraCoumaric

acid

50200 μM

Ferulic acid

0.25 mM

Hydroxytyrosol

40100 μM

25 mg/kg daily

[97]

[98]

[99]

[100]

[101]

[102]

Table (Contd.)

induction of ER stress; increase of Beclin 1 and Atg5 expres

sion; induction of apoptosis

activation of AMPK and SIRT1 kinases

destabilization of mitochondria membranes; enhance

ment of ROS production

inhibition of mTOR and phosphorylation of S6 kinase

enhancement of ROS production; increase of p62 con

tent, activation of MAPK; induction of apoptosis

decrease of expression of Atg7, Beclin 1, LC3B, level of

FOXO3 mRNA

induction

activation

induction

disruption

decrease4

A375, 451Lu

HepG2

N2a

murine hepatocytes, HeLa

PC3, DU145

rat skeletal muscles

Notes: Cell lines: glioma (U138, U251, U373, U373MG), lymphocytic leukemia (CEMC115, CEMC714, IM9, Jurkat, Molt4), melanoma

(451Lu, A375, B16, G361), myelogenous leukemia (HL60, K562, RAW264.7), neuroblastoma (N2a), squamous cell oral carcinoma (U87,

U87MG, YD10B), breast cancer (MCF7, MDAMD231, SUM159), gingival cancer (OECM1), stomach cancer (AGS, MKN28), lung

cancer (A549), pleural mesothelioma cancer (ACCMESO1), bladder cancer (T24), osteosarcoma (U2OS), liver cancer (Bel7402,

HepG2, Huh7, SMMC7721), pancreatic cancer (MIA PaCa2), prostate cancer (DU145, LNCaP, PC3), colon cancer (CacoH2, COLO

201, HCT116, HT29), cervical cancer (HeLa), tongue cancer (SAS), ovarian cancer (A2780, CaOV3, ES2, OVCAR3); cardiomyoblasts

(H9c2), embryonic kidney cells (293T, HEK293, HEK293T), preadipocytes (hASCs), murine fibroblasts (MEF), embryonic fibroblasts

(NIH/3T3), aortic endothelial cells (BAEC), umbilical vein endothelial cells (HUVEC).

1Rapamycin and starvationinduced autophagy.

2Glucoseinduced autophagy.

3In combination with indole3carbinol.

4Physical exerciseinduced autophagy.

306 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

direct interaction: for example, in vitro investigation of

more than 100 kinases identified 14 that demonstrated

more than twofold drop in activity when resveratrol was

added at a concentration of 20 μM [64]. In some studies,

induction of autophagy by resveratrol was related to ROS

production; furthermore, the antioxidant Nacetylcys

teine cancelled the effect [62]. The increased transloca

tion of transcription factors Nrf2, FOXO1, FOXO3A, and

FOXO4 that can enhance autophagy into the nucleus was

also shown [108, 119].

Another widespread phytoalexin – pterostilbene –

demonstrates more pronounced antitumor and Nrf2

inducing activity in comparison with resveratrol, which

can be related to its higher lipophilicity and metabolic

stability [73, 120]. Pterostilbene is an important bioac

tive component of grapes and blueberries, but it is almost

absent in grape wines as it degrades rapidly under light in

the presence of oxygen. Clinical studies established that

consumption of pterostilbene at a dose of 250 mg/day for

68 weeks is safe [121]. It was also shown that astrin

genin (3,3′,4′,5tetrahydroxystilbene) inhibited ROS

generation, activation of spleen tyrosine kinase and

MAPK8, as well as autophagy in murine macrophages

and the RAW264.7 macrophage cell line under the

action of oxidized LDL [122]. Yet another stilbene –

pinosylvin (3,5dioxystilbene) – at rather high concen

tration (100 μmol/liter) induced apoptosis and

autophagy in bovine aortic endotheliocytes via activa

tion of caspase3 and AMPK [123]. The synthetic ana

log (3,4,4′trihydroxystilbene) demonstrated higher

antioxidant and protective activity in comparison with

resveratrol in multiple experimental systems [124], and

it also induced autophagy in the A549 lung cancer cell

line via enhancement of ROS generation and inhibition

of mTOR [125].

Flavonoids are the largest group of natural polyphe

nolic compounds. Two aromatic rings A and B linked by

a threecarbon bridge forming pyran and pyrone cycle,

i.e. heterocycle (a C6–C3–C6sequence) comprise its

structural elements. More than 10,000 flavonoids have

been isolated from plants and characterized, and it is like

ly that their number is significantly higher because

flavonoid composition of many plant species is still

unknown. The majority of flavonoids can also be consid

ered as derivatives of chromane and flavan. Depending on

the flavonoid skeleton structure, flavonols, flavones, cat

echins, anthocyanidins, isoflavones, leucoanthocyani

dins, dihydroflavonols, chalcones, and aurones can be

distinguished [2]. Trivial names are common for

flavonoids that are predominantly connected to the first

isolation of the compound. For example, bark of the

Quercus velutina L. oak is the source of quercetin (Fig. 2),

wood of the Douglas fir (Pseudotsuga taxifolia) – source

of taxifolin (dihydroquercetin), and the Baikal skullcap

(Scutellariae baicalensis) – source of baicalein. Quercetin

occurs widely in plants (apples, onion) and is produced in

a form of tablets, capsules, and aqueous solutions, while

not being registered as a medication for treatment of any

disease in humans. Genistein (Fig. 2) is a principle

flavonoid in soya beans and soyabased products, and

because of structural similarity with 17βestradiol it

exhibits estrogenic activity and is capable of binding

estrogen receptors ERαand ERβ.

Numerous studies have shown that in many experi

mental and biological systems flavonoids exhibit anti

radical and antioxidant actions, and they are very active

with respect to radicals emerging either in lipid or aque

ous phases and inhibit processes of lipid peroxidation

both during the initiation stage interacting with ROS

(О2

•–, OH•, 1O2, HOCl, H2O2) and during the chain

propagation stage acting as hydrogen atom donors for

lipid radicals [2]. Structural analysis and experimental

data indicate a direct relationship between the antioxi

dant efficiency of flavonoids and the number of phenol

OH groups in their molecules. Experimental studies in

aqueous systems revealed the following structural ele

ments of flavonoid molecules that are most important

for antiradical activity: (i) availability of catechol struc

ture in the B ring; (ii) double bond between carbon

atoms 2 and 3 preferably together with a carbonyl group

in the position C4, and (iii) OH group in the position C5

together with C4 carbonyl group [2]. The ability of

flavonoids to induce the Keap1/Nrf2/ARE signaling sys

tem and, therefore, protect cells under oxidative stress

conditions, was shown in the majority of studies [126].

Due to the large number and diversity of flavonoids,

many of them have not been investigated with respect to

autophagy initiation. The autophagy initiation by

quercetin was shown to be dosedependent and reached

its maximum in HeLa cells at a flavonoid concentration

of 50 μM [87], and in gastric cancer cells MKN28 it

increased linearly up to concentration 160 μM [84]. The

ability of quercetin to accumulate in mitochondria was

shown, which was accompanied by membrane depolar

ization and enhanced production of ROS [38]. The

induction of ER stress by flavonoids results in the release

of Ca2+ and activation of a series of kinases inducing

autophagy [94, 97].

Catechins are important components of the popular

drink – tea, which is produced from the leaves of the

shrub Camellia sinensis. Phenolic compounds constitute

up to ∼3040% of the dry weight of nonfermented green

tea with catechin derivatives (catechin, epicatechin, epi

gallocatechin, epicatechin3gallate, and epigallocate

chin3gallate) being the major ones. The ester of epigal

locatechin and gallic acid – epigallocatechin3gallate

(Fig. 2) – is the principle catechin (more than 50%) of

green tea, which is very popular in China and Japan, and

it is present in lesser amounts in red tea (oolong variety)

and in yellow tea, and even in lesser amounts in black tea,

which is favored by Europeans and Americans [2]. Tea is

very popular among the nations of all continents, and

PLANT PHENOLS AND AUTOPHAGY 307

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

numerous facts establishing its antitumor, cardioprotec

tive, antiviral, and antibacterial properties have accumu

lated.

Analysis of inhibition of DPPH radicals and super

oxide anion radical by the teaderived polyphenolic com

pounds revealed a direct relationship between the number

of OH groups and antiradical activity: epigallocatechin

3gallate >epicatechin3gallate >epigallocatechin >

epicatechin >catechin [2]. Despite the fact that in major

ity of experimental systems catechins inhibit radicals less

effectively in comparison with flavonols and flavones

because they do not contain conjugated double bonds in

the C3structure, formation of esters with gallic acid

enhances their antioxidant activity significantly.

Hormesis of action of catechins, especially the action of

epigallocatechin3gallate, was noted for different exper

imental systems, as it is easily oxidized with generation of

ROS as well as reducing iron ions [127]. Epigallocat

echin3gallate is an effective inducer of Nrf2; it operates

through direct interaction with the Keap1 cysteine

residues or through enhancement of ROS production,

which activates kinases and inhibits Keap1 [128].

Epigallocatechin3gallate at low concentrations (10

20 μM) induced and enhanced endotoxinstimulated

autophagy, while at high concentrations (2001000 μM) it

activated caspases and initiated apoptosis [79]. In addi

tion to antioxidant action, epigallocatechin3gallate

affects the activity of multiple transcription factors: it

inhibits NFκB, AP1, STAT1, STAT3, FOXO1, and

activates Nrf2 [129]. Many epigallocatechin gallates

increase the calcium ion concentration in cytosol, which

results in activation of AMPK and enhancement of the

production of NO•by calmodulindependent NOsyn

thases [129].

Vitamin E is a fatsoluble factor required for repro

duction in rats that can be found at high concentrations in

wheat germ oils and salad seeds. It was discovered in 1922

by American scientists Herbert McLean Evans and

Katharine Scott Bishop. In experiments, this factor pre

vented development of infertility in rats that were fed only

by a mixture of casein, lard, milk fat, salt, and yeast. It

was possible to restore reproductive function by supple

menting their food ration with salad leaves or wheat germ

oil, while the addition of fish oil or flour did not result in

any improvement. The compounds related to vitamin E

comprise a group of chroman6ol derivatives (6hydrox

ychroman) – tocopherols different in the degree of

methylation and position of methyl groups in the chro

man nucleus. Tocopherols and tocotrienols are the most

important compounds that differ in position of methyl

substituents in the chroman nucleus and the number of

double bonds in the side chain (Fig. 3).

Antiradical activity is demonstrated by all toco

pherols and tocotrienols in different in vitro experimental

systems [2], while these compounds do not exhibit pro

nounced inducing activity with respect to the

Keap1/Nrf2/ARE signaling system [11]. On the other

hand, α, γ, and δtocotrienols activated the Keap1/

Nrf2/ARE system and induced expression of the ARE

regulated genes in estrogenreceptor negative breast can

cer cell line MDAMB231 [130]. Tocotrienols and α

tocopherol succinate demonstrated pronounced

autophagyinducing ability [74, 131]. Unlike αtoco

pherol, which did affect viability of rat pancreatic cells up

Fig. 3. Structures of major vitamin E homologs.

Tocotrienols

Tocopherols αanalog

βanalog

γanalog

δanalog

308 ZENKOV et al.

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

to a concentration of 800 μM, low concentrations of the

palm oil tocotrienols (1020 μM) induced apoptosis and

autophagy [132]. This suggested that the main target of

tocotrienol action in cells were mitochondria, damage to

which resulted in the release of Beclin 1, cytochrome c,

and enhancement of ROS production initiating apoptosis

and autophagy [133]. Subsequent studies revealed other

mechanisms of tocotrienol action. For example, γ

tocotrienol triggered ER stress in breast cancer cells,

which activated PERK and IRE1 kinases [75]. The possi

bility of inhibition of a number of kinases (PI3K, p38

MAPK, Akt) was also shown [74, 131].

Phenolic acids are abundant in plants: simple acids

(hydroxybenzoic, protocatechuic, vanillic, and others)

can be found almost everywhere, among the hydroxycin

namic acids (series C6C3) the four most abundant ones

(oxycinnamic, caffeic, ferulic, and sinapic) (Fig. 2) are

biogenetic precursors for the majority of other phenolic

compounds [2]. Hydroxycinnamic acids are in the com

position of complex compounds (lignins, lignans,

hydrolyzable tannins, etc.), and, hence, can be formed

during their degradation in an organism. The compounds

of this group can also be formed in animal tissues during

metabolism of polyphenols such as flavonoids [2].

Investigation of the action of ferulic acid (Fig. 2) indi

cates that while its antioxidant activity shows up at low

concentrations (1200 μM), the ability to induce

autophagy appears only at concentrations above 1 mM

[100].

Hydroxytyrosol is an important component of olives

and olive oil comprising 7080% of the total phenolic

fraction. Hydroxytyrosol is readily oxidized to the

quinone form (Fig. 4) due to the availability of the cate

chol structure, which makes it an effective antioxidant

capable of inhibition of the radical forms of ROS and for

mation of complexes with metals of variable valency. The

ability of hydroxytyrosol to induce the expression of

Nrf2regulated genes was shown for different cell types;

moreover, the catechol structure was also found impor

tant for activation of the Keap1/Nrf2/ARE signaling sys

tem [134]. It is likely that due to unique redox and antiox

idant properties of hydroxytyrosol its effect on autophagy

is ambiguous: it induces formation of autophagosomes,

but inhibits their degradation by lysosomes [101].

The natural polyphenols are now suggested as an

effective treatment for a broad spectrum of diseases: car

diovascular [117, 135, 136], neurodegenerative [7, 127,

137], oncological [8, 138], inflammatory diseases [139],

diabetes, metabolic disorders, obesity [98, 109, 119, 140,

141], and many others. Moreover, autophagy is an impor

tant target of plant polyphenol action. Despite the variety

of mechanisms of the action of phenols, several principle

mechanisms can be identified.

1) Change of intracellular redox balance due to

mitochondrial membrane depolarization or selfoxida

tion of phenols, which results in enhancement of ROS

production [38]. Many autophagy proteins contain cys

teine residues that are prone to oxidation, with some of

them being already identified as in mTOR (C2460 and

C2467), Atg4 (C81), and PARKIN protein (C59, C95,

C182) [6]. Similar cysteine residues were found in tumor

suppressor p53 (C173, C235, C239), transcription factors

NFκB (p50) (C62), AP1 (C154, C269), and HIF1α

(C800), and in a series of protein kinases participating in

the autophagy induction [142]. That is why autophagy

enhancement observed under the action of phenols can

probably be a reaction to the development of intracellular

oxidative stress [143].

2) mTOR is an important target of the action of plant

phenols; it can be inactivated either directly or indirectly

via AMPK and PI3K/Akt [17, 144]. In particular, cur

cumin at low concentrations (2.520 μM) causes dissoci

ation of Raptor and mTOR in the mTORC1 complex,

which results in its inactivation [145]. Resveratrol

enhances an inhibiting effect of another component of

the complex – DEPTOR – on mTOR [146].

3) Change of cell energy balance occurs with ser

ine/threonine AMPK being its principle sensor [147,

148]. From the structural point of view, AMPK comprise

threeprotein complexes consisting of a catalytic αsub

unit (1αor 2α), regulatory βsubunit (1βor 2β), and

AMPbinding subunit (γ1, γ2, or γ3) [148]. AMP and

ADP are classical AMPK activators; Ca2+ and H2O2also

activate the kinase [147]. AMPK activation inhibits

mTORC1 through phosphorylation of both the Raptor

protein in composition of the mTORC1 complex as well

as the TSC2 protein, which reduces its stabilizing effect

on mTORC1; furthermore, many natural phenols acti

vate AMPK [41, 49, 51, 129, 140].

4) Among the seven sirtuins identified in mammals

(SIRT1SIRT7, histone deacetylases that transfer acetyl

groups to histones and thus regulate DNA transcription

activity), SIRT1 directly participates in autophagy induc

tion [149]. The activated SIRT1 deacetylates AMPK and

enhances its activity as well as activates the FOXO3 tran

scription factor that controls transcription of genes of the

Atg proteins, catalase, and MnSOD [9, 115]. Although

many plant polyphenols can activate SIRT1, it has been

shown in multiple studies that resveratrol has the highest

activity [115].

5) Many transcription factors are targets of the

action of phenolic compounds. The most direct relation

Fig. 4. Redox transformations of hydroxytyrosol.

PLANT PHENOLS AND AUTOPHAGY 309

BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

ships with autophagy were revealed for the factors of the

NFE2 Nrf1 [150] and Nrf2 [6, 16] subfamily. The Nrf1

transcription factor is controlled directly by the mTORC1

complex, and the Keap1 repressor of the Nrf2 factor

binds the Atg8/LC3 complex through the p62 protein,

which is quite reasonable because all these structures play

an important protective role during oxidative stress.

The majority of current studies have been conducted

with cell cultures, since investigation of autophagy in vivo

is quite difficult. That is why much work lies ahead before

researchers can provide any practical recommendations

on the possibility to regulate autophagy by plant phenols.

This work was financially supported by the Russian

Foundation for Basic Research (project No. 1400

00551a).

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Metabolic and Antioxidant Responses of Different Control Methods to the Interaction of Sorghum sudangrass hybrids-Colletotrichum boninense

Article

Full-text available

Aug 2024
INT J MOL SCI

Colletotrichum boninense is the main pathogenic fungus causing leaf spot disease in Sorghum sudangrass hybrids, which seriously impairs its quality and yield. In order to find an efficient and green means of control, this study used the agar disk diffusion method to screen for a fungicide with the strongest inhibitory effect on C. boninense from among several bacteria, fungi, and chemicals. Then, the changes in the plant’s antioxidant system and metabolic levels after treatment were used to compare the three means of control. The lowest inhibitory concentration of Zalfexam was 10 mg/mL, at which point C. boninense did not grow, and the inhibition rates of Bacillus velezensis (X7) and Trichoderma harzianum were 33.87–51.85% and 77.86–80.56%, respectively. Superoxide dismutase (SOD) and chitinase were up-regulated 2.43 and 1.24 folds in the Trichoderma harzianum group (M group) and SOD activity was up-regulated 2.2 folds in the Bacillus velezensis group (X7 group) compared to the control group (CK group). SOD, peroxidase (POD), and chitinase activities were elevated in the Zalfexam group (HX group). The differential metabolites in different treatment groups were mainly enriched in amino acid metabolism and production, flavonoid production, and lipid metabolism pathways. Compared with the diseased plants (ZB group), the M, X7, HX, and CK groups were co-enriched in the tryptophan metabolic pathway and glutamate–arginine metabolic pathway, and only the CK group showed a down-regulation of the metabolites in the two common pathways, while the metabolites of the common pathways were up-regulated in the M, X7, and HX groups. In addition, the salicylic acid–jasmonic acid pathway and ascorbic acid–glutathione, which were unique to the M group, played an important role in helping Sorghum sudangrass hybrids to acquire systemic resistance against stress. This study fills the gap in the control of Colletotrichum boninene, which causes leaf spot disease in Sorghum sudangrass hybrids. This paper represents the first reported case of biological control for leaf spot disease in Sorghum sudangrass hybrids and provides a reference for the control of leaf spot disease in Sorghum sudangrass hybrids as well as other crops infected with Colletotrichum boninense.

Study on Differences of Metabolites among Different Ganoderma Species with Comprehensive Metabolomics

Research

Full-text available

Jul 2024
J. Fungi

In traditional Chinese medicine, Ganoderma is a kind of edible and medicinal mushroom, which is widely used because of its significant pharmacological activity. There are many species within the Ganoderma genus, each with different material bases and applications. However, detailed studies on these species are still lacking. In this study, we investigated the metabolites of G. leacontextum (B), G. lucidum (C), G. tsugae (S) from Changbai Mountain, and G. tsugae (M) from Mongolia using metabolomics. The PCA results indicated minimal differences between M and S, whereas B and S exhibited significant variations. A total of 708 differential metabolites were identified in this study, with steroids, triterpenoids, phenols, and quinones being the major metabolites. Specifically, triterpenoids and steroids were higher in C. Meanwhile, phenolic compounds were more abundant in B. Additionally, quinones were more abundant in M and S. We validated some of the main compounds, and the results showed that paracetamol was most abundant in B, making paracetamol a potential marker for identifying B. Additionally, vitamin K3 was found to be more abundant in M and S, which can serve as a marker for their identification. This study provides new insights and a theoretical basis for the development and utilization of the genus Ganoderma.

Autophagy characteristics of phytoestrogens in management and prevention of diseases: A narrative review of in-vivo and in-vitro studies

Article

Full-text available

Jun 2023

Phytoestrogens are non-steroid polyphenolic materials present in 300 plants. Regarding their structural similarities to estradiol, phytoestrogens attach to estrogen receptors and display anti-or pro-estrogenic activities. This review explored phytoestrogens’ potential advantages and autophagy properties in light of their future application for disease management, highlighting how phytoestrogens could modulate autophagy. Research has examined the prospective benefits of phytoestrogens for the anticipation and management of various conditions, including signs of menopause, tumors, skin deterioration, osteoporosis, heart disease, neurodegenerative conditions, disorders of the immune system, and metabolic syndrome, owing to their therapeutic effects. As phytoestrogens can activate or inhibit autophagy, which has antioxidant, apoptotic, anti-mutagenic, anticancer, transcriptional, and genomic impacts on cancer and aging illnesses, phytoestrogens could influence diseases through the modulation of autophagy. The collaborative research on animal models, utilization of genetic techniques, and administration of pharmacolog¬ically active substances has indicated the possible therapeutic benefits of autophagy modulation in various illnesses. Further research is required to illustrate the pathways by which phytoestro¬gens modulate autophagy and the possible therapeutic effects on these diseases.

Induction of Human Oral Squamous Carcinoma Apoptosis by Derris scandens Benth and Elephantopus scaber Linn Extracts

Article

Full-text available

Jun 2022
NAT PROD COMMUN

D scandens ( Derris scandens Benth.) and E scaber ( Elephantopus scaber Linn.) contain flavonoids and phenolic acids, which have antitumor activity in various cancer cell lines. Oral cancer is among the most common cancers in Southeast Asia, and the survival rate remains low. Thus, this study screened 2 ethanolic plant extracts for cytotoxicity on the oral human squamous carcinoma cell line (HSC-2), and compared the mechanisms of action. Extracts of D scandens and E scaber showed cytotoxicity against HSC-2 cells in a dose-dependent manner. Observation of nuclear morphology by Hoechst 33342 staining revealed chromatin condensation. Apoptosis was confirmed by Annexin V-FITC staining and cell sorting (fluorescence-activated cell sorting) analysis. We demonstrated that cancer apoptosis was accompanied by changes in the expression of procaspase 3 and that D scandens-mediated apoptosis in HSC-2 cells was potentiated by protein kinase B (Akt) and B-cell lymphoma-2 (Bcl-2), while E scaber apoptosis was mediated by mitogen-activated protein kinase (MAPK) pathways, involving stress-activated protein kinases/jun amino-terminal kinase (SAPK/JNK) and p38-MAPK. Further investigation into targets for apoptosis induction by these plant extracts may have potential in oral cancer therapy.

Mitochondrial Quality and Quantity Control: Mitophagy Is a Potential Therapeutic Target for Ischemic Stroke

Article

Full-text available

May 2022
MOL NEUROBIOL

Ischemic stroke is a cerebrovascular disease with high mortality and disability, which seriously affects the health and lives of people around the world. Effective treatment for ischemic stroke has been limited by its complex pathological mechanisms. Increasing evidence has indicated that mitochondrial dysfunction plays an essential role in the occurrence, development, and pathological processes of ischemic stroke. Therefore, strict control of the quality and quantity of mitochondria via mitochondrial fission and fusion as well as mitophagy is beneficial to the survival and normal function maintenance of neurons. Under certain circumstances, excessive mitophagy also could induce cell death. This review discusses the dynamic changes and double-edged roles of mitochondria and related signaling pathways of mitophagy in the pathophysiology of ischemic stroke. Furthermore, we focus on the possibility of modulating mitophagy as a potential therapy for the prevention and prognosis of ischemic stroke. Notably, we reviewed recent advances in the studies of natural compounds, which could modulate mitophagy and exhibit neuroprotective effects, and discussed their potential application in the treatment of ischemic stroke.

A Decade of Mighty Lipophagy: What We Know and What Facts We Need to Know?

Article

Full-text available

Nov 2021
OXID MED CELL LONGEV

Lipids are integral cellular components that act as substrates for energy provision, signaling molecules, and essential constituents of biological membranes along with a variety of other biological functions. Despite their significance, lipid accumulation may result in lipotoxicity, impair autophagy, and lysosomal function that may lead to certain diseases and metabolic syndromes like obesity and even cell death. Therefore, these lipids are continuously recycled and redistributed by the process of selective autophagy specifically termed as lipophagy. This selective form of autophagy employs lysosomes for the maintenance of cellular lipid homeostasis. In this review, we have reviewed the current literature about how lipid droplets (LDs) are recruited towards lysosomes, cross-talk between a variety of autophagy receptors present on LD surface and lysosomes, and lipid hydrolysis by lysosomal enzymes. In addition to it, we have tried to answer most of the possible questions related to lipophagy regulation at different levels. Moreover, in the last part of this review, we have discussed some of the pathological states due to the accumulation of these LDs and their possible treatments under the light of currently available findings.

Ecophysiological, transcriptomic and metabolomic analyses shed light on the response mechanism of Bruguiera gymnorhiza to upwelling stress

Article

Aug 2024

Genetic modulators of neurodegenerative diseases and their amelioration by natural products

Chapter

Jan 2023

Regression-Classification Algorithm for Screening of Antiradical Activity of Flavonoids and the Related Structures

Article

Sep 2022

A review of biologically active flavonoids as inducers of autophagy and apoptosis in neoplastic cells and as cytoprotective agents in non‐neoplastic cells

Article

May 2022

Mark D'Arcy

Phytochemicals are a diverse group of compounds found in various fruits, vegetables, nuts, and legumes. Many phytochemicals have been observed to possess health benefits. Some have been found to be chemoprotective or can act as chemotherapeutics by inducing autophagy, apoptosis, or otherwise regulating the cell cycle. Many also act as potent antioxidants. Flavonoids are a subclass of bioactive phytochemicals consisting of two phenolic benzene rings, joined together by a heterocyclic pyran or pyrone. It has been observed in multiple studies that there is a correlation between diets rich in flavonoids and a reduction in cancer levels, heart disease, neurodegenerative diseases, and other pathologies. As foods containing flavonoids are widely consumed, and their mechanisms of action are still only partially understood, this review was compiled to compare the effects and mechanisms of action of some of the most widely characterized and publicized flavonoids. The flavonoids silibinin, quercetin, isorhamnetin, luteolin, curcumin genkwanin, and acacetin, together with flavonoid extracts from papaw and Tephroseris kirilowii (Turcz) Holub, a member of the Daisy family, were found to be potent regulators of the cell cycle. The decision to overview these specific flavonoids was based on their therapeutic effects, and/or their potential effects. The sparsity of data comparing these flavonoids was also a key consideration. These flavonoids all modulated to some extent the pathways of autophagy and/or apoptosis and regulated the cell cycle, inflammation, and free radical levels. This explains why they are protective of healthy or moderately damaged cells, but toxic to neoplastic or pre‐cancerous cells.

B19, a Novel Monocarbonyl Analogue of Curcumin, Induces Human Ovarian Cancer Cell Apoptosis via Activation of Endoplasmic Reticulum Stress and the Autophagy Signaling Pathway

Article

Full-text available

Aug 2013
INT J BIOL SCI

Background: The unfolded protein response, autophagy and endoplasmic reticulum (ER) stress-induced apoptosis regulate tumor cell fate and have become novel signaling targets for the development of cancer therapeutic drugs. Curcumin has been used to treat several different cancers, including ovarian cancer, in clinical trials and research; however, the role of ER stress and autophagy in the therapeutic effects of curcumin and new curcumin analogues remains unclear. Methods: Cell viability was determined using the MTT assay. Apoptosis was detected using flow cytometry with PI/Annexin V-FITC staining. The expression levels of ER stress- and autophagy-related proteins were analyzed by western blotting. The activation of autophagy was detected using immunofluorescence staining. Results: We demonstrated that B19 induced HO8910 cell apoptosis in a dose-responsive manner. We also determined and that this effect was associated with corresponding increases in a series of key components in the UPR and ER stress-mediated apoptosis pathways, followed by caspase 3 cleavage and activation. We also observed that B19 treatment induced autophagy in HO8910 cells. The inhibition of autophagy using 3-methyladenine (3-MA) increased levels of intracellular misfolded proteins, which enhanced ovarian cancer apoptosis. Conclusions: Our data indicate that ER stress and autophagy may play a role in the apoptosis that is induced by the curcumin analogue B19 in an epithelial ovarian cancer cell line and that autophagy inhibition can increase curcumin analogue-induced apoptosis by inducing severe ER stress.

The Role of AMPK Pathway in Neuroprotection

Chapter

Full-text available

Oct 2016
ADV EXP MED BIOL

Adenosine monophosphate-activated kinase (AMPK) is a highly conserved protein kinase found in all eukaryotic genomes. It exists as heterotrimeric protein consisting of α, β, and γ subunits. AMPK is activated by elevated levels of adenosine mono-phosphate (AMP), which is produced during conditions of low ATP production and perhaps mitochondrial dysfunction. Activation of AMPK has been shown to regulate a large number of downstream pathways. These will either increase energy production such as increase oxidation of fatty acids and glucose, or decrease energy utilization such as inhibiting synthesis of glycogen, fatty acid synthesis, and protein synthesis. In addition, being a key regulator of physiological energy dynamics, AMPK has been demonstrated to play roles in regulating various cellular processes such as mitochondrial biogenesis (Jager et al. Proc Natl Acad Sci U S A 104:12017–12022, 2007), autophagy (Hyttinen et al. Rejuven Res 14:651–660, 2011) and inflammation and immune responses (Giri et al. 2004). Retinal neurons have a high energy demand but have a poor energy storage capacity. Because of this, it is likely that the AMPK signaling pathway plays an important role in maintaining energy balance, and therefore may be a therapeutic target to prevent or delay retinal degeneration.

Protective Macroautophagy Is Involved in Vitamin E Succinate Effects on Human Gastric Carcinoma Cell Line SGC-7901 by Inhibiting mTOR Axis Phosphorylation

Article

Full-text available

Jul 2015
PLOS ONE

Vitamin E succinate (VES), a potential cancer therapeutic agent, potently induces apoptosis and inhibits the growth of various cancer cells. Autophagy has been supposed to promote cancer cell survival or trigger cell death, depending on particular cancer types and tumor microenvironments. The role of autophagy in the growth suppressive effect of VES on gastric cancer cell is basically unknown. We aimed to determine whether and how autophagy affected the VES-induced inhibition of SGC-7901 human gastric carcinoma cell growth. SGC-7901 cells were treated with VES or pre-treated with autophagy inhibitor, chloroquine (CQ) and 3-methyladenine (3-MA). Electron microscopy, fluorescence microscopy and Western blot were used to study whether VES induced autophagy reaction in SGC-7901 cells. Western blot evaluated the activities of the mammalian target of rapamycin (mTOR) axis. Then we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry to detect the level of cell viability and apoptosis. Collectively, our data indeed strongly support our hypothesis that VES treatment produced cytological variations that depict autophagy, increased the amount of intracellular green fluorescent protein-microtubule associated protein 1 light chain 3 (GFP-LC3) punctate fluorescence and the number of autophagic vacuoles. It altered the expression of endogenous autophagy marker LC3. VES activated the suppression of mTOR through inhibiting upstream regulators p38 MAPK and Akt. mTOR suppression consequently inhibited the activation of mTOR downstream targets p70S6K and 4E-BP-1. The activation of the upstream mTOR inhibitor AMPK had been up-regulated by VES. The results showed that pre-treatment SGC-7901 with autophagy inhibitors before VES treatment could increase the capacity of VES to reduce cell viability and to provoke apoptosis. In conclusion, VES-induced autophagy participates in SGC-7901 cell protection by inhibiting mTOR axis phosphorylation. Our findings not only strengthen our understanding of the roles of autophagy in cancer biology, but may also be useful for developing new treatments for gastric cancer patients.

Natural Compounds Modulating Mitochondrial Functions

Article

Full-text available

Jun 2015
EVID-BASED COMPL ALT

Mitochondria are organelles responsible for several crucial cell functions, including respiration, oxidative phosphorylation, and regulation of apoptosis; they are also the main intracellular source of reactive oxygen species (ROS). In the last years, a particular interest has been devoted to studying the effects on mitochondria of natural compounds of vegetal origin, quercetin (Qu), resveratrol (RSV), and curcumin (Cur) being the most studied molecules. All these natural compounds modulate mitochondrial functions by inhibiting organelle enzymes or metabolic pathways (such as oxidative phosphorylation), by altering the production of mitochondrial ROS and by modulating the activity of transcription factors which regulate the expression of mitochondrial proteins. While Qu displays both pro- and antioxidant activities, RSV and Cur are strong antioxidant, as they efficiently scavenge mitochondrial ROS and upregulate antioxidant transcriptional programmes in cells. All the three compounds display a proapoptotic activity, mediated by the capability to directly cause the release of cytochrome c from mitochondria or indirectly by upregulating the expression of proapoptotic proteins of Bcl-2 family and downregulating antiapoptotic proteins. Interestingly, these effects are particularly evident on proliferating cancer cells and can have important therapeutic implications.

Hydroxytyrosol Promotes Superoxide Production and Defects in Autophagy Leading to Anti-proliferation and Apoptosis on Human Prostate Cancer Cells

Article

Jul 2013

Hydroxytyrosol, an important polyphenolic compound found in olive oil, has shown anti-tumor activity both in vitro and in vivo. However, effects of hydroxytyrosol on prostate cancer are largely unkown. We found that hydroxytyrosol preferentially reduces the viability of human prostate cancer cells (PC-3, DU145) compared to an immortalized non-malignant prostate epithelial cell line (RWPE-1). Exposure of PC-3 cells to 80 mu mol/L hydroxytyrosol resulted in significant increases in both superoxide production and activation of apoptosis. These increases were accompanied by mitochondrial dysfunction, defects in autophagy, and activation of MAP kinases. Moreover, N-acetyl-cysteine (NAC), an efficient reactive oxygen species (ROS) scavenger, was able to reverse the hydroxytyrosol-induced effects of cell viability loss, defects in autophagy, and activation of apoptosis. This evidence suggests that ROS play a vital role in the loss of PC-3 cell viability. However, MAPK inhibitors including U0126 (for Erk1/2), SB203580 (for p38MAPK) and SP600125 (for JNK) did not decrease hydroxytyrosol-induced growth inhibition, suggesting that these kinases may not be required for the growth inhibitory effect of hydroxytyrosol. Moreover, addition of ROS scavengers (i.e. NAC, catalase, pyruvate, SOD) in the growth media can prevent hydroxytyrosol induced cell viability loss, suggesting that extracellular ROS (superoxide and hydrogen peroxide) facilitate the anti-proliferation effect of hydroxytyrosol in prostate cancer cells. The present work firstly shows that hydroxytyrosol induces apoptotic cell death and mitochondrial dysfunction by generating superoxide in PC-3 cells. This research presents preliminary evidence on the in vitro chemopreventive effect of hydroxytyrosol, and will contribute to further investigation of hydroxytyrosol as an anti-cancer agent.

Toward an understanding of the role of a catechol moiety in cancer chemoprevention: The case of copper- and o -quinone-dependent Nrf2 activation by a catechol-type resveratrol analog

Article

Aug 2015
MOL NUTR FOOD RES

Catechol moieties are commonly present in dietary natural products that exert cancer chemopreventive activity. While the oxidative conversion of catechols into their corresponding o-quinones is generally considered to contribute to their cancer chemopreventive effects, the mechanism of the intracellular conversion has not been fully elucidated. Among resveratrol and its hydroxylated analogs examined, only 3,4-dihydroxy-trans-stilbene exerted cytoprotective effects against t-butylhydroperoxide-induced death of HepG2 cells. This resveratrol analog activated the Nrf2 pathway through stimulating phosphorylation of Akt and inducing keap1 modification, thereby resulting in its nuclear translocation and subsequent transcriptional induction of phase II detoxifying enzymes. Its cytoprotective effect through Nrf2 activation was largely abrogated by pretreatment of cells with DTT, a sulfhydryl-containing nucleophile, and neocuproine, a specific chelating agent for copper ions. We identified 3,4-dihydroxy-trans-stilbene as a novel Nrf2 activator which is converted intracellulary into its corresponding o-quinone electrophile by Cu2+ ions. The copper-mediated oxidation was required for the Nrf2 activation, subsequent transcriptional induction of phase II detoxifying enzymes and ultimately for cytoprotection. The findings demonstrate a previously under-recognized role for intracellular copper ions in the cancer chemopreventive effects of catechol-containing dietary natural products. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The critical role of quercetin in autophagy and apoptosis in HeLa cells

Article

Aug 2015
TUMOR BIOL

In recent years, the effects of quercetin on autophagy and apoptosis of cancer cells have been widely reported, while effects on HeLa cells are still unclear. Here, HeLa cells were subjected to quercetin treatment, and then proliferation, apoptosis, and autophagy were evaluated using MTT, flow cytometry, and MDC staining, respectively. The LC3-I/II, Beclin 1, active caspase-3, and S6K1 phosphorylation were detected using Western blot assay. The ultrastructure of HeLa was observed via transmission electron microscope (TEM). Our findings showed that quercetin can dose-dependently inhibit the growth of HeLa cells. The MDC fluorescence was enhanced with increased concentration of quercetin and hit a plateau at 50 μmol/l. Western blot assay revealed that LC3-I/II ratio, Beclin 1, and active caspase-3 protein were enforced in a dose-dependent method. However, the phosphorylation of S6K1 gradually decreased, concomitant with an increase of autophagy. In addition, TEM revealed that the number of autophagic vacuoles was peaked at 50 μmol/l of quercetin. Besides, interference of autophagy with 3-MA led to proliferation inhibition and increased apoptosis in HeLa cells, accompanied by the decreased LC3-I/II conversion and the increased active caspase-3. In conclusion, quercetin can inhibit HeLa cell proliferation and induce protective autophagy at low concentrations; thus, 3-MA plus quercetin would suppress autophagy and effectively increased apoptosis.

Correction: KEAP1-NRF2 signalling and autophagy in protection against oxidative and reductive proteotoxicity

Article

Aug 2015
BIOCHEM J

Maintaining cellular redox status to allow cell signalling to occur requires modulation of both the controlled production of oxidants and the thiol-reducing networks to allow specific regulatory post-translational modification of protein thiols. The oxidative stress hypothesis captured the concept that overproduction of oxidants can be proteotoxic, but failed to predict the recent finding that hyperactivation of the KEAP1-NRF2 system also leads to proteotoxicity. Furthermore, sustained activation of thiol redox networks by KEAP1-NRF2 induces a reductive stress, by decreasing the lifetime of necessary oxidative post-translational modifications required for normal metabolism or cell signalling. In this context, it is now becoming clear why antioxidants or hyperactivation of antioxidant pathways with electrophilic therapeutics can be deleterious. Furthermore, it suggests that the autophagy-lysosomal pathway is particularly important in protecting the cell against redox-stress-induced proteotoxicity, since it can degrade redox-damaged proteins without causing aberrant changes to the redox network needed for metabolism or signalling. In this context, it is important to understand: (i) how NRF2-mediated redox signalling, or (ii) the autophagy-mediated antioxidant/reductant pathways sense cellular damage in the context of cellular pathogenesis. Recent studies indicate that the modification of protein thiols plays an important role in the regulation of both the KEAP1-NRF2 and autophagy pathways. In the present review, we discuss evidence demonstrating that the KEAP1-NRF2 pathway and autophagy act in concert to combat the deleterious effects of proteotoxicity. These findings are discussed with a special emphasis on their impact on cardiovascular disease and neurodegeneration. © 2015 Authors; published by Portland Press Limited.

Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

Article

Jul 2015
ACTA PHARMACOL SIN

To investigate the effects of 3,4,4'-trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro. Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy. Treatment with 3,4,4'-THS (10-80 μmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4'-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 μmol/L). Moreover, 3,4,4'-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4'-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)Conclusion:3,4,4'-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4'-THS-induced apoptosis of A549 cells, thus combination of 3,4,4'-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.

Natural Nrf2 activators in diabetes

Article

Jul 2015

Prediabetes and diabetes is rising worldwide. Control of blood glucose is crucial to prevent or delay diabetic complications that frequently result in increased morbidity and mortality. Most strategies include medical treatment and changes in lifestyle and diet. Some nutraceutical compounds have been recognized as adjuvants in diabetes control. Many of them can activate the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which has been recognized as a master regulator of the antioxidant response. Recent studies have described the role of Nrf2 in obesity, metabolic syndrome, nephropathy, retinopathy and neuropathy, where its activation prevents the development of diabetes and its complications. It has been demonstrated that natural compounds derived from plants, vegetables, fungi and micronutrients (such as curcumin, sulforaphane, resveratrol and vitamin D among others) can activate Nrf2 and, thus, promote antioxidant pathways to mitigate oxidative stress and hyperglycemic damage. The role of some natural Nrf2 activators and its effect in diabetes is discussed. Copyright © 2015. Published by Elsevier B.V.