Steroid plant hormones: Effects outside plant kingdom

Steroid plant hormones: Effects outside plant kingdom

Steroids 97 (2015) 87–97 Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Review Steroid plan...

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Steroids 97 (2015) 87–97

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

Review

Steroid plant hormones: Effects outside plant kingdom Vladimir N. Zhabinskii ⇑, Natalia B. Khripach, Vladimir A. Khripach Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich St., 5/2, 220141 Minsk, Belarus

a r t i c l e

i n f o

Article history: Received 9 July 2014 Received in revised form 12 August 2014 Accepted 25 August 2014 Available online 9 September 2014 Keywords: Brassinosteroids Medicinal aspects Phytohormones Anticholesterolemic activity Anticancer activity Antiviral activity

a b s t r a c t Brassinosteroids (BS) are the first group of steroid-hormonal compounds isolated from and acting in plants. Among numerous physiological effects of BS growth stimulation and adaptogenic activities are especially remarkable. In this review, we provide evidence that BS possess similar types of activity also beyond plant kingdom at concentrations comparable with those for plants. This finding allows looking at steroids from a new point of view: how common are the mechanisms of steroid bioregulation in different types of organisms from protozoa to higher animals. Ó 2014 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on fungi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on protozoa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects in warm-blooded animals and medical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Toxicology and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Anticholesterolemic action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Anticancer effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Anabolic and adaptogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Antiviral effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Other effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Steroids have been recognized as the hormones of higher vertebrates for quite a long time, more than half a century [1]. In the

Abbreviations: BS, brassinosteroids; CC50, 50% cytotoxic concentration; EBl, 24-epibrassinolide; EC50, half maximal effective concentration; HSV, herpes simplex virus; HBl, 28-homobrassinolide; MPP+, 1-methyl-4-phenylpyridinium; TNF-a, tumor necrosis factor a. ⇑ Corresponding author. Tel./fax: +375 172 678 647. E-mail address: [email protected] (V.N. Zhabinskii). http://dx.doi.org/10.1016/j.steroids.2014.08.025 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

87 88 89 89 89 89 89 90 91 92 93 93 94 94 94

middle of the sixties it became evident that steroids play a hormonal role in invertebrates also, in particular in the moulting functions of insects and other arthropods [2]. At about the same time steroid hormones of fungi were found [3]. Isolation of brassinolide and a number of related compounds (named as brassinosteroids), having hormonal functions in plants [4], showed that steroids are versatile hormonal regulators, characteristic to most organisms inhabiting the earth. A rapid progress in the study of steroidal plant hormones resulted in establishing many intimate details of their action in plants and led to their use in agriculture as crop increasing and

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plant-protecting agents [5]. The development of such agents implied detailed toxicological studies of BS, including their influence on bees, aqueous organisms and animals. As was expected, BS proved to be non-toxic compounds [6–9]. However, it was not the only result of these studies. Many experiments revealed a pronounced adaptogenic effect of BS to non-plant test organisms. This offered an incentive to investigate thoroughly brassinosteroid effects outside plant kingdom. Their knowledge will contribute to a solution of a more general problem, namely ‘‘How steroidal hormones, typical of certain organisms, relate to the functioning of organisms that belong to other classes or kingdoms?’’. One has to be aware that no complete answer can be given to this question at the present stage of research. The obtained data are still fragmentary and subject to criticisms. One among them is that many studies have been conducted using synthetic BS analogues of unnatural structure. It creates some limitations for their use and generalization in respect to real steroidal plant hormones where brassinolide 1 (Fig. 1) is recognized to play a central role [10,11]. Brassinolide itself has practically never been used for biological experiments on non-plant organisms, although a number of more available natural BS (e.g., epibrassinolide 2, homobrassinolide 3 and corresponding 6-ketones 4 and 5) were investigated quite extensively. Among many BS analogues those containing a (22S,23S)-diol function (e.g., 6 and 7) should be mentioned as being of a considerable interest in these studies. Although plant growth promoting activity of (22S,23S)-analogues is very low [12–14], in

S R S

R S

S

2 2

S

S

S

Fig. 1. Structures of compounds 1–8.

(R)

(R)

some tests on non-plant organisms these easily available compounds revealed remarkable effects [15–21]. 2. Effects on insects Structural considerations were probably the main reason why studies of BS action outside the plant kingdom were started on insects, moulting hormones of which (e.g., ecdysterone 8, Fig. 2) are very close structurally to BS. The first experiments showed that steroidal phytohormones could affect normal growth and development of insects. A number of BS effects were revealed at different levels [22], including intact animals [15,23,24], isolated tissues [23,25,26], cultured cells [27,28], particular insect neurons [29], and protein molecules (ecdysteroid receptors) [23,28,30,31]. However, the results of these experiments are not always consistent with each other. Thus, a number of BS were tested in in vitro experiments on imaginal discs isolated from fly species Phormia terrae-novae and Calliphora vicina and exhibited only a slight (if any) agonistic ecdysteroid activity and a significant antagonistic dose dependent effect when concomitantly applied with ecdysterone 8 [22]. Other studies showed BS acting as either agonists [27] or antagonists [25,30], and none of BS tested in the Drosophila melanogaster BII cell bioassay revealed either agonist or antagonist activity [32]. Feeding the cockroach Periplaneta americana with artificial diet containing (22S,23S)-homobrassinolide 3 resulted in a lengthening the larval stage by moulting delay [15], although closely related (22S,23S)-homocastasterone 5 proved to be inactive in this assay. BS were toxic to the larvae of the cotton leafworm Spodoptera littoralis when applied by injection in high doses at the end of the last instar [23]. The observed result could not be attributed to interference of BS in the moulting process since the effects from BS application differed from those of ecdysterone 8 or its non-steroidal agonist. The investigation in Phormia terrae-novae [25] showed that BS could compete with ecdycteroids for the invertebrate nuclear steroid hormone receptor EcR, and this was later confirmed by other studies [22–24,29,30,33]. However, the affinity in most experiments was 10- to 1000 fold lower than that observed for binding to radiolabeled ponasterone A, and no competition at all was observed for EcR in intact Se4 cells even at relatively high (100 lM) concentration of EBl [28]. A number of synthetic hybrids of BS and ecdysteroids were prepared and assessed for their activities in the Drosophila melanogaster BII cell bioassay [33]. Nearly all tested compounds displayed no ecdysteroid agonist activity demonstrating the high specificity for the EcR receptor. A distinct activity was noticed only for the hybrid 9 (Fig. 2), however, it was still 2000-fold less active than ecdysterone 8. Similar studies were performed with two castasterone/ponasterone A hybrid compounds [34]. The (22R)-isomer 10 was more potent than the corresponding (22S)-isomer for the competitive inhibition of [3H]ponasterone incorporation (about 100 times with Kc cells and about 35 times with Sf-9 cells). In general, to date, experimental evidence confirming cross reactivity between steroidal insect and plant hormones is lacking.

( S) ( S)

Fig. 2. Structures of compounds 8–10.

(R)

(R)

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Based on the current knowledge of both hormonal systems [35], they are likely evolved and functioning independently. The observed effects on insects may be the result of BS cytotoxic properties [28]. The highest content of BS in natural sources was found in pollen collected from flowers [4]. That means that BS have been consumed by nectar- and pollen eating insects (bees, in particular) over many years of co-evolution and could become for them food essential components. A number of beneficial effects from administration of EBl to bees could be regarded as experimental confirmation of this idea [36–39]. Thus, feeding the bees with sugar syrup laced with EBl resulted in an increase in their lifespan up to 100% [36] and stimulated queens to more intensive oviposition [36,37,39]. In another study, no changes in the dynamic of oviposition onset were registered [40].

3. Effects on fungi It was repeatedly shown that application of BS resulted in an enhanced resistance of plants to fungal pathogens [41]. However, interpretation of the obtained results should be made with caution. The observed on vegetative plant effects should be evaluated in the context of BS action on the entire plant–pathogen system instead of being considered as an indication of their direct antifungal properties [42,43]. In most cases, a pronounced stimulative effect was observed on treatment of fungi with BS [42,44–46]. Thus, the growth of mycelia of the fungus Psilocybe cubensis was two to three times faster under the influence of 10 2 ppm of (22S,23S)-homobrassinolide 7 in comparison with untreated control [44]. BS treatment led also to earlier appearing of the first flush of fruit bodies and to the increase of dry mass. Some BS were found to be promising for industrial production of mushrooms Agaricus bisporus and Pleurotus ostreatus [47,48].

4. Effects on fishes Intensive studies of BS effects on fishes started in the second half of the 1990s [49] in Russia and within a short period of time have led to a practical application of the research outcomes [50– 56] in fish farming for the protection of embryos, larvae and fingerlings from unfavorable environmental ecological conditions and for increasing fish production [57]. The first experiments were carried out with Russian sturgeon Acipenser gueldenstaedti belonging to a unique group of bony fish. Sturgeon fingerlings treated with epibrassinolide (EBl) solution (10 4 mg/L) prior to their exposure to toxicants (such as CuSO4, phenol, or the detergent) were significantly less negatively influenced by the toxicants than untreated control [49]. This could be seen from the higher abilities of fingerlings with regard to movements, reactions to a sonic signal, resistance to a current and training. Similar results were obtained for Black Sea salmon, carp, crucian and silver carp [53]. Analysis of physiology-biochemical parameters of treated and control fishes showed that EBl possessed antioxidant properties [49] and stabilized hematoencephalic and histohematogenous barriers [58,59]. Prolonged exposure of silver carp to copper or organic toxicants resulted in an increase in erythrocyte catalase activity. Prior application of EBl returned it to nearly control values [60]. Immersing Siberian sturgeon in an EBl solution led to an increase of ceruloplasmin level (over 500% higher on the fifth week of the study) [51]. This is an indication of EBl immunostimulatory properties which might have resulted through the effect of activated leukocytes on hepatocytes. A significant decrease of hemoglobin content in the blood was observed under the action of the toxicants. This parameter was greatly improved in EBl-treated fishes [49].

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Lipid peroxidation is the process of oxidative degradation of lipids that becomes more intense under stress conditions. BS were shown to decrease in plants the accumulation of malonic dialdehyde [61], which is the most important product of lipid degradation. The same tendency was observed in fishes exposed to copper, phenol or detergent toxicants [53]. Level of malonic dialdehyde in fishes treated by EBl and toxicants showed no statistical difference with the control (toxicant-untreated) group. At the same time, in the toxicant-treated group without EBl level of malonic dialdehyde was significantly higher. A pronounced effect of BS on fish reproduction was found [55,62–64]. Thus, treatment of Russian sturgeon eggs with EBl gave a significant increase of a fecundation, hatching and larvae survival [62]. The EBl-treated eggs produced the fingerlings with better morphological characteristics and resistance to stress. Immersing sturgeon fish larvae in EBl solution led to better survival of the fingerlings and to increase their body weight. The same effects were also seen on phytophagous fishes (grass carp and silver carp) [63,64]. Treatment with EBl of spermatozoons of Russian sturgeon enhanced their activity and viability, especially in the case of spermatozoons reactivated after cryoconservation [55].

5. Effects on protozoa A significant concentration-dependent effect of epibrassinolide was revealed on infusoria Tetrahymena pyriformis in the culture medium [65]. Among a wide range of investigated concentrations, two of them (with epibrassinolide content in culture medium 410 7 and 410 13 mg/ml) were found to be the most efficient in the increasing population growth and adaptation coefficient.

6. Effects in warm-blooded animals and medical aspects The finding of the protective and growth-activating effects of BS in fishes provided impetus for a systematic search of BS-initiated responses in other vertebrates and particularly in warm-blooded animals. The first results were obtained in rodents in the course of toxicological studies [66]. They showed the ability of BS to influence the reproductive sphere [67], steroid hormonal balance [68,69], some biochemical [70] and physiological [71,72] parameters that reflected clear tendency to stimulative and adaptive shifts in experimental animals. Similarity of BS action in plants and in non-plant organisms raised the awareness of the potential value of these compounds for medicinal applications and initiated intensive studies. Some of the results are reviewed in recent publications [73–76], and these developments will be mentioned only briefly in this review. 6.1. Toxicology and pharmacokinetics These studies confirmed the safety conclusions of the earlier experiments [6–9] and extended the borders for harmless BSapplication. In acute experiment, toxicity of EBl was characterized by value LD50 above 5000 mg/kg after its oral administration to mice [77]. EBl demonstrated no mutagenic properties in Ames’ test (S. typhimurium, TA100) [78]. The presence of EBl in a system of metabolic activation had no influence on DNA damage rate by benzidine and electrophoretic mobility both native and damaged DNA of lambda phage [79] thus demonstrating the lack of genotoxicity for BS. Intracutaneous injection of EBl to white mice caused no significant delayed-type allergic hypersensitivity responses [80]. Based on lack of maternal and embryo-fetal toxicity in Wistar rats, HBl was concluded to be nonteratogenic at doses as high as up to 1000 mg/kg body weight [81].

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Table 1 Effect of EBl on total blood serum cholesterol (mg/deciliter) under intragastrular administration during 36 weeks. Control

68.11 ± 4.75

Dose of EBl 0.2 lg/kg

2 lg/kg

20 lg/kg

200 lg/kg

62.17 ± 5.54 ( 9%)

57.81 ± 6.34 ( 15%)

54.25 ± 3.17 ( 20%)

51.08 ± 5.15 ( 25%)

The pharmacokinetics of EBl was studied in rats by intragastric administration of its 3H-labelled form [82]. It was well absorbed from the gastrointestinal tract following the administration and quickly distributed to blood, liver, intestines, lungs and kidneys. The serum highest radioactivity was reached in 30 min after administration. The serum half-life was about 3 h after administration. Similarly, the highest activity in liver also took place after 30 min and then it gradually decreased. The accumulation of 3HEBl (and/or its metabolites) went slower in kidneys, where its highest level could be seen after 6 h. The quickest EBl-accumulating organ was found to be small intestine, where only 15 min were needed to reach its highest concentration. Since significant amount of 3H-EBl and (or) products of its biotransformation were found in kidneys, urine and faeces of experimental animals, it was concluded that these are the major ways for its elimination from the body. 6.2. Anticholesterolemic action Plant sterols and their derivatives are known as inhibitors of intestinal cholesterol absorption and agents for lowering the plasma total and LDL cholesterol levels [83–85]. As an oxidized form of plant sterols, BS could be expected to possess similar activity, although there are contradictory data about the influence of oxysterols on the development of atherosclerosis [86–88]. Promising results were obtained in studies on the effects of BS on cholesterol level [89]. Application of EBl to rats with normal blood cholesterol level fed with a normal diet in daily doses of 2–200 lg/kg for 36 weeks gave 9–25% lower cholesterol depending on a dose in a manner, where higher doses corresponded to a higher cholesterol lowering effect (Table 1). In rats fed with high-cholesterol diet, the intake of a daily dose of 2 lg/kg of EBl for 4 weeks reduced the plasma concentration of total cholesterol for 34% and triglycerides for 58% in comparison with control animals that received the same diet without EBl (Table 2). In EBl-fed animals, plasma concentration of vitamin A and vitamin E increased for 16% and 53%, correspondingly, in comparison with the control. In rats fed with high-cholesterol diet, the

intake of a daily dose of 20 lg/kg of EBl for 4 weeks reduced the plasma concentration of total cholesterol for 44%, triglycerides for 68% and low-density lipoprotein for 11% in comparison with control animals that received the high-cholesterol diet only. In EBl-fed animals, plasma concentration of high-density lipoprotein, vitamin A and vitamin E was higher than in the control for 47%, 30% and 51% correspondingly. A considerable enhancement of redox-vitamins level reflects a decrease of oxidative stress and can contribute in this way to anti-atherosclerosis action of EBl. A similar trend towards decreasing cholesterol level on BS application was also observed in humans [90,91]. A group of volunteers (10 people) with hypercholesterolemia was assigned to consume daily 15 lg of EBl (Table 3) [90]. Participants experienced a decrease in total serum cholesterol from initial elevated values of 5.70–4.73 mmol/L, which is in the normal range. Analysis of the lipid profile showed that the observed changes were to a greater extent due to the reducing the content of LDL fraction from 4.03 to 2.97 mmol/L. Another study was undertaken with subjects with a normal level of cholesterol [91]. Both the control and experimental group consisted of 30 healthy volunteers. Each person from the experimental group daily received 15 lg of EBl during 1 month. Before and immediately after finishing the experiment all volunteers were subjected to the complex investigation involving basic laboratory tests. There were no significant differences in hematological and biochemical parameters between the two groups except for the level of cholesterol. Thus, statistically significant decrease of cholesterol and triglycerides levels was noted (38% for cholesterol and 41% for triglycerides). It is an interesting question about the origin of all these effects. Steroids are known to exhibit both genomic and non-genomic effects [92,93]. It is difficult to expect any specific genomic response from BS having in mind that until now these compounds have never been found in human, animals, or insects. However, a possibility of a non-specific response remains. Recently it has been shown that a number of nuclear receptors play an important role in maintaining the proper level of cholesterol in the body [94]. A nuclear receptor LXR induces ABC1 reverse transporter of Table 3 Effect of EBl on lipid metabolism in humans after four-week administration.

Cholesterol, mmol/L Triglycerides, mmol/L HDL, mmol/L VLDL, mmol/L LDL, mmol/L Atherogenic index

Normal range

Control

EBl, 15 lg daily

3.2–5.2 0.49–2.0 1.03–1.52 <2.6 <3.9 <3

5.70 ± 0.67 0.37 ± 0.08 1.50 ± 0.08 0.17 ± 0.04 4.03 ± 0.56 2.77 ± 0.27

4.73 ± 0.67 0.67 ± 0.08 1.44 ± 0.04 0.30 ± 0.04 2.97 ± 0.68 2.28 ± 0.42

Table 2 Effect of EBl on lipid metabolism and levels of redox-vitamins in blood serum of rats under high-cholesterol diet (four-week administration). Control

High-cholesterol diet

High-cholesterol diet and EBl 2 lg/kg

High-cholesterol diet and EBl 20 lg/kg

47.12 ± 2.77

98.20 ± 3.96

Triglycerides, mmol/L

0.56 ± 0.01

1.90 ± 0.38

VLDL, %

7.78 ± 0.52

4.89 ± 0.35

LDL, %

66.16 ± 0.65

75.55 ± 0.49

HDL,%

25.05 ± 0.55

19.57 ± 0.34

Vitamin A, mmol/L

0.60 ± 0.01

0.23 ± 0.02

Vitamin E, mmol/L

2.79 ± 0.02

1.24 ± 0.08

64.94 ± 5.15 ( 34%) 0.80 ± 0.09 ( 58%) 5.31 ± 0.24 (+9%) 75.24 ± 1.23 (0%) 19.45 ± 1.14 ( 1%) 0.26 ± 0.02 (+16%) 1.90 ± 0.14 (+53%)

55.04 ± 4.36 ( 44%) 0.60 ± 0.04 ( 68%) 4.33 ± 0.45 ( 11%) 66.97 ± 1.23 ( 11%) 28.72 ± 1.22 (+47%) 0.29 ± 0.01 (+30%) 1.88 ± 0.10 (+51%)

Total cholesterol, mg . deciliter

1

In brackets: percentage related to the high-cholesterol diet.

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cholesterol that pumps out cellular cholesterol, resulting in lowering dietary cholesterol. Another nuclear receptor FXR activates cytochrome P450 hydroxylase CYP7A1 that converts excess of cholesterol to bile acids. Certain oxygenated steroids and particularly products of cholesterol oxidation (oxysterols) act as the signaling molecules that bind to LXR/FXR proteins and stimulate transcription of the corresponding genes. It cannot be excluded that EBl (being a highly oxygenated sterol) interferes with the process that leads to the diminishing of cholesterol level in blood. It is very likely that another possible mode of BS anticholesteremic action could be similar of that of Lovastatin [95] which also belongs to natural isoprenoids and has in its molecule lactone ring, a feature which is also characteristic for BS. In this case, BS would act as an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), which catalyzes the rate limited step in cholesterol biosynthesis – reduction of 3-hydroxy-3-methylglutarylCoA by NADPH to mevalonate. Cholesterol-decreasing properties of EBl could be also a part of its adaptogenic effect, which is found in animals and in plants and realized in the latter ones, at least partly, via regulation of the fluidity and permeability of membranes and the activity of membrane associated proteins. Such kinds of activities have been documented for BS in plants [96,97]. In spite of lack of definite data on mechanism of BS anticholesteremic action, they can be used in the development of cholesterolcontrolling and arteriosclerosis-preventing agents [89,98]. 6.3. Anticancer effects Steroids have been known as a source of novel leads in the development of therapeutics for the treatment of cancer [99]. A number of side chain oxygenated sterols isolated from plants and marine organisms are toxic to mammalian cells, especially in fast proliferating tumor cells [100]. It was natural to expect similar activity from BS most of which are 22,23-oxygenated steroids. Some studies showed promising results for cancer therapy and have been patented by several teams of researchers [101–105]. A first step in this direction was done in [106] where mouse hybridoma cells were grown in culture media containing 10 16–10 9 mol/L of EBl. The treated cells showed an increase in G0/G1 phase and decrease in S phase. In addition, a drop in intracellular antibody level and an increase in the value of mitochondrial membrane potential were noticed. The next step was the study of BS cytotoxic effects. In a Calcein AM cytotoxicity assay castasterone showed a slight activity against CEM and RPMI 8226 cell lines [107,108]. More systematic studies were carried out with EBl and homocastasterone on the breast (MCF-7/MDA-MB-468) and prostate (LNCaP/DU-145) cancer cell lines [109]. Both compounds were shown to inhibit cell growth of the cancer cells. BS treatment resulted in arrest of cell cycle in the G1 phase and an induction of apoptosis [104,110]. A range of techniques including flow

cytometry, Western blotting, TUNEL, DNA ladder assays and immunofluorescence analyses was used for study of BS-induced apoptosis of human prostate cancer cell lines LNCaP and DU-145 [111]. Cell growth inhibition and G1 cell cycle arrest were accompanied by reductions in cyclin D1, CDK4/6 and pRb expression. Treatment of DU-145 cells with BS led to an increase of cells in the G2/M phase and down-regulation of cyclins A and B1. Apoptotic effects of brassinolide on human prostate cancer PC-3 cells was shown to be associated with caspases-3 activation [112]. EBl-induced apoptosis of LNCaP and DU-145 cancer cells was accompanied by a decrease of intracellular polyamine levels and a significant down-regulation of ornithine decarboxylase [113]. Structure activity relationship studies of natural BS and their close analogues revealed certain structural features needed for cytotoxic activity [75,109]. The highest activity was observed for compounds such as castasterone and homocastasterone having a 6-oxo functionality and a (24S)-side chain. The corresponding 6oxo-7-oxalactones or (24R)-derivatives were less active. It was shown also that 2a,3a-diol group is important for cytotoxicity. A series of studies was undertaken for the purpose of evaluating the effects of 22,23-dihydroxystigmastane derivatives (natural BS and analogues) [114–116]. The highest cytotoxicity effect against human breast carcinoma MCF-7, human ovary carcinoma CaOv, and human prostate carcinoma LnCaP cells was observed for compounds 11 and 12 (Fig. 3) containing an equatorial hydroxyl group at C-3 [116]. The most polar compounds (including 28-homobrassinolide 3 and 28-homocastasterone 5) showed the lowest activity. It was found that for every pair of isomers, (22R,23R)-derivatives were significantly more toxic than their (22S,23S)-counterparts. The observed difference was assumed to be due to the spatial structure of the side chain. Computational search showed that (22S,23S)-side chain accepted many various conformations whereas about 96% of (22R,23R)-diols existed in just few related energy minima. In this way, the higher cytotoxicity of (22R,23R)22,23-dihydroxystigmastane derivatives was explained by their more rigid side chain. Similar results were obtained for derivatives with a campestane, ergostane [117] and cholestane [118] carbon skeletons. Thus, (22R,23R)-diols 13 and 14 were more cytotoxic (IC50 1.6–1.8 lM) for MCF-7 cells in comparison with the corresponding (22S,23S)isomers (IC50 >49 lM) [117]. Low-polar diols 15 and 16 were found to be the most efficient among tested (22R,23R)-22,23-dihydroxycholestanes including 28-norcastasterone and 28-norbrassinolide [118]. Incubation of human prostate adenocarcinoma cells with compounds of this series (IC50 = 13–28 lM) resulted in blocking cell proliferation and inducing apoptosis (23–33%). Some compounds induced also arrest of the cell cycle in the S- and G2/Mphases. A structurally close to BS (22R)-22-hydroxy-5a-cholestan-3,6-dione 17 from brown alga Cystoseira myrica was found to exhibit the pronounced cytotoxicity against human liver

(R) (R)

(R)

Fig. 3. Structures of compounds 11–17.

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OH HO

HO

CF3 F 3C F

H N

H2N

HO

H

18

O

S

N

H

O

O 19 CO2H

HO

N

H

O

O

20

HO

H

O

O 21

Fig. 4. Structures of compounds 18–21.

(HEPG-2) and colon (HCT116) cancer cells (with IC50 2.96 and 12.38 lM, respectively) [119]. Several attempts were tried to improve cytotoxic activity by structural modifications of the steroidal molecule (Fig. 4). A norcastasterone hepta-fluorinated derivative 18 was the only analogue of this type which exhibited a slight cytotoxicity (IC50 = 35.3 lM against CEM cell line) [120]. A distinct cytotoxicity (IC50 = 7–15.8 lM) against MGC 7901 (human gastric carcinoma), HeLa (human cervical carcinoma) and SMMC 7404 (human liver carcinoma) cells displayed cholestane lactones 19 and 20 [121]. A number of monohydroxylated BS analogues with a carboxylic group in the side chain (e.g., lactone 21) were tested for antiproliferative activity against human normal fibroblasts and cancer cell lines (T-lymphoblastic leukemia CEM, breast carcinoma MCF7, cervical carcinoma cell line HeLa) [122]. However, none of them displayed any detectable effect. A comparison of the anticancer and the brassinolide-type activity of the fluoro analogues [123] showed no correlation: while ergostane derivatives were most active in the anticancer, the corresponding androstane derivatives were the best in the bean second-internode bioassay. Antiangiogenic properties of BS were found to be another type of their activity that is potentially useful in cancer treatment [105,124]. Angiogenesis is known to be an important process in the development of cancer as malignant cells depend on an adequate supply of oxygen. Inhibition of this process represents a promising target for antitumor therapy [125]. Out of 21 tested BS, EBl and homocastasterone at a concentration of 30 lM reduced migration of HUVEC cells to 59% and 40%, respectively [124]. An inhibition of tube formation was noticed for a number of BS, including brassinolide, homobrassinolide, and epicastasterone. Evidently, structural features of BS are not very relevant for this kind of activity since synthetic analogues 22 [124] and 23,24 [105] (Fig. 5) inhibited angiogenesis more effectively than natural BS. Compounds 23 and 24 were also patented as anti-inflammatory and antiviral agents for the treatment of epidemic keratoconjunctivites and herpetic stromal keratis [126]. Biotransformation of xenobiotics serves as an important defense mechanism for the body. Toxic compounds are converted into less reactive and polar substances that can easily be excreted. Sometimes, however, this process results in the generation of more harmful metabolites [127]. A classic example is the P450-mediated activation of benzo(a)pyrene. The formation of its phenolic and diol derivatives is the main pathway by which this procarcinogenic polycyclic aromatic compound is eliminated from the body. On the other hand, epoxidation of the corresponding dihydroxy derivatives yields compounds exhibiting strong carcinogenic properties. BS were shown to affect the monooxygenase activity of liver microsomes [128–130]. A strong inhibitory effect of BS on benzo(a)pyrene oxidation was observed for (22S,23S)-homobrassinolide 3 and (22S,23S)-homocastasterone 5. The corresponding

HO

H

O

22 OH HO

O

23 OH HO

O

24

Fig. 5. Structures of compounds 22–24.

natural BS showed only a weak activity. It should be noted that no significant effect on the benzo(a)pyrene hydroxylation (what is necessary for elimination of this compound) was observed. 6.4. Anabolic and adaptogenic effects Anabolic and adaptogenic properties of ecdysteroids is a wellknown phenomenon [131,132]. It is not surprising, therefore, that the corresponding experiments were performed with BS inspired by the structural likeness of both types of hormones. Administration of HBl (20–60 mg/kg) was found to have multiple anabolic effects on rats, including increase of food intake, body weight gain, lean body mass, and gastrocnemius muscle mass [17]. Application of BS resulted also in an improved physical fitness, in particular, significant increases in treadmill performance and enhanced grip strength were achieved in rats by administration of HBl [133]. EBl at doses of 2–20 mg/kg improved the static efficiency and swimming physical endurance in mice [134]. An increase of tolerance of mammalian organisms to various stresses was noticed on EBl administration [135]. Anabolic properties of BS were used to yield a higher meat and milk productivity of farm animals and meat producing broilers [136] and for increasing the fertility of the bull sperm-producer [137]. In contrast to the usual anabolic androgenic steroids, which act through binding to the intracellular androgen receptor (and which have a lot of adverse side effects on people), BS seem to exert their

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OH

OH

HO R1

R 2O

25: R1=Br, R2=OH 26: R1=OH, R2=OH 27: R1=OH, R2=F

HO F

HO

28

Fig. 6. Structures of compounds 25–28.

action in a different way. Thus, HBl revealed a low androgenic activity in the Hershberger assay and no significant binding to the androgen receptor in vitro [17]. The activation of PI3K/Akt signaling pathway was suggested as a possible explanation of the BS action that followed from an increased Akt phosphorylation in vitro under BS action [18]. 6.5. Antiviral effects A search for antiviral effects of steroids was started in the 1990s, first among progestagens, glucocorticoids and dehydroepiandrosterone [138]. The ability of BS to enhance resistance of plants to the viral pathogens [4,139] offered an incentive to look for similar properties outside plant kingdom. Starting from the 2000s, an impressive study in this area was performed by researchers from Argentina [73,74,140]. A large number of BS (both natural and synthetic analogues) of stigmastane series was prepared and tested for antiviral activity against animal viruses: poliovirus [73], herpes simplex viruses HSV-1 [141–147] and HSV-2 [143], measles virus [16], vesicular stomatitis virus [148] and the arenaviruses [142,144,149]. The relative effectiveness of BS analogues in inhibiting viral replication compared to inducing cell death is measured by their selectivity index (ratio CC50/EC50). Most of the studied compounds exhibited a good activity against the tested viruses, with the selectivity index higher than that of parent homocastasterone [138]. It was found that analogues with a (22S,23S)-diol moiety revealed a better activity compared to the corresponding (22R,23R)-diols. The presence of an electronegative group (fluorine or hydroxyl) at C-5 also favored high antiviral activity. Thus, BS analogues 25–27 (Fig. 6) were active against all tested viruses [73]. EC50 values for compounds 25 and 27 against measles virus were 4 and 3 lM, respectively, with selectivity indexes of 44 and 27 (higher than for reference drug ribovirin) [16]. The 3b-fluroanalogue analogue 28 displayed even better EC50 values of 1 lM against measles virus, but it proved to be too cytotoxic. In search for a possible mechanism of antiviral action, influence of 25 on viral protein synthesis in HSV-1 infected Vero cells was examined [145]. It was found no effects on early events of the virus multiplication cycle, but the late protein synthesis was strongly inhibited by the presence of 25. This mechanism is different from the one of antiviral medications acyclovir and foscarnet, that was confirmed by studies of the effects of their combinations with 25 [146]. A synergistic increase in the antiviral activity of acyclovir (29.3%) and foscarnet (47.2%) was observed in the presence of 25. An in vivo study of the antiherpetic properties of 25 in the murine stromal keratin experimental model led to the conclusion that the compound did not exert a direct antiviral effect [144]. Instead, it acted as an inductor or an inhibitor of cytokine production, thus modulating the response of epithelial and immune cells to herpes virus infection [150]. The protective effect in mice was explained as a balance between immunostimulatory and immunosuppressive effects of 25. An inhibitory effect of 25 and 26 on the TNF-a production (an excess of which contributes to autoimmune diseases) can be considered as another evidence of immunomodulating

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properties of the studied compounds [151]. This inhibitory effect may be linked, at least in part, with the ability of both compounds to reduce the incidence of herpetic stromal keratitis in infected mice (although none of them revealed any anti-HSV activity in vivo) [144]. A marked protective effect of BS was observed against human immunodeficiency virus infection [152]. The in vitro treatment with EBl increased significantly the cell lifetime. The amount of the living cells in the infected culture treated with EBl was more than 50% higher in comparison with untreated control at 4–5th days after infecting. Moreover, a significantly decreased production of viral-specific antigens on the cell surface was observed at 3rd day after infecting. 6.6. Other effects HBl subchronic exposure in rats was shown to have a strong influence on glucose homeostasis [153–156]. Experimental animals had a significant rise in the serum insulin level and decrease in the blood sugar. In addition, HBl-treated rats exhibited an elevated hexokinase activity in brain, heart, liver, kidney, and testis. It was speculated that HBl played a role of a transcriptional activator of hexokinase gene, promoting enhanced hexokinase mRNA synthesis in vivo in rat tissues. Another evidence of anti-diabetic properties of BS was obtained in the experiments with fat dietinduced obese mice [19]. HBl chronic administration (50 mg/kg daily for 8 weeks) reduced hyperglycemia and improved oral glucose tolerance. This treatment reduced the expression of key gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) and increased phosphorylation of AMPactivated protein kinase in the liver tissue. Structure–activity relationship studies showed that a 6-keto group was more preferable for achieving high glucose metabolism-modulating activity in comparison with typical for BS 6-keto-7-lactone function [19]. It is worth of mentioning that BS-induced lowering blood glucose level was associated with their anabolic effects [17]. This is another evidence of similarity in the actions between BS and ecdysteroids. The latter were demonstrated to affect glucose metabolism and insulin sensitivity in animals also [132]. The protective properties of BS on lipid peroxidation and antioxidative system in plants is a well-known phenomenon [157]. It seemed interesting to study similar effects of BS on non-plant organisms. Hyperglycemia is known to be associated with the oxidative stress and lipid peroxidation. Increased content of endogenous malondialdehyde and 4-hydroxy-2-nonenol is considered as lipid peroxidation indices. Level of these products in normal and diabetic rats was significantly suppressed by the treatment with EBl [158]. Increased activity of catalase enzyme and enhanced content of glutathione evidenced an EBl-induced elevated antioxidant defence. Another consequence of oxidative stress is DNA damage. An attempt was made to study antigenotoxic activity of extracts from Centella asiatica against H2O2-induced DNA damage in human blood lymphocytes [159]. A fraction of the extract containing castasterone (10 9 M) was effective in diminishing the DNA damage by 89%. 1-Methyl-4-phenylpyridinium (MPP+) is a potent inducer of oxidative stress in dopaminergic neurons and is used as an in vitro cellular model of Parkinson’s disease. Neuronal PC12 cells could be efficiently rescued by the pretreatment with EBl (10 9 M) from MPP+-induced cellular death [160]. EBl reduced the production of intracellular reactive oxygen species and modulated activities of superoxide dismutase, catalase, and glutathione peroxidase. Inhibition of MPP+-induced apoptosis was attributed to reducing the DNA fragmentation as well as the Bax/Bcl-2 protein ratio and cleaved caspase-3. Structure–function relationship

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studies showed that 6-ketones exhibited nearly the same neuroprotective activity as EBl [20]. Topical administration of (22S,23S)-homobrassinolide 7 was shown to reduce significantly wound size and accelerate wound healing in mice [21,161,162]. The observed effect was explained by a positive modulation of the inflammatory and re-epithelialization phases of the skin wound repair process as a result of enhancing Akt signaling at the edges of the wound and (in vitro) enhancing migration of fibroblasts in the wounded area. EBl was patented as a means for anti-wrinkle cosmetics and rough skin treatment [163]. It enhanced formation of collagen in both human dermal fibroblast and epidermal cells. There is an indication that BS can be used in treating androgenassociated conditions, such as benign prostatic hyperplasia and androgenic alopecia [164]. The observed effect was attributed to the inhibition of 5a-reductase activity, which took place in EBltreated human foreskin and bovine prostatic tissues. A significant reduction of the prostate weight in old male rats was explained by modulation of the androgen receptor by EBl [165].

7. Conclusions Brassinosteroids are the first group of steroid-hormonal compounds isolated from and acting in plants. Among numerous physiological effects of BS growth stimulation and adaptogenic activities are especially remarkable. Nowadays, there are many evidences that BS produce the same types of activity also beyond plant kingdom when applied at concentrations comparable with those for plants. In our book [4] we summarized all available data on effects of BS outside plant kingdom. Even that time, the accumulated results reflected effects of BS in all other kingdoms in addition to plants: fungi, protista, monera and animals. Unfortunately, they were sporadic, sometimes conflicting and very poor to make certain conclusions. In fact, there were practically no data on BS effects in higher animals except the results on toxicity. During the time that has elapsed since the publication of this book, a lot of new data appeared concerning the action of plant steroid hormones in non-plant organisms, particularly in animals and in humans. Most of them confirm the similarity of adaptation-stimulating properties of BS in plants and the outside plant kingdom. These data contribute to a new understanding of steroidal hormones as versatile bioregulators of all living creatures [166]. Relatively young evolutional age of vertebrates and higher animals suggests the possible importance for them of bioregulating mechanisms and their key mediators that have appeared earlier and nowadays exist in plants and some lower organisms. In comparison with animals, plants have a more ancient origin and their regulatory systems, which developed during evolution into the highly specialized hormonal systems of human and higher animals, could logically be expected to be more universal. Therefore the possibility exists that their hormonal substances might have bio-regulatory functions in younger organisms standing higher on the evolutionary ladder. From biochemical point of view, BS having ‘‘the most economic’’ structure of hydroxylated sterols (that means relatively simple biosynthetic pathway to these hormones from normal plant sterols), would seem to be very close structurally to the bioregulating steroids of the most ancient organisms (like some marine polyoxysteroids), and that is why they could participate in basic steroid signalling pathways, which have been inherited by younger organisms standing higher on the evolutionary ladder. The comparability of active doses of BS in plants, animals, fungi, protista, and monera, and similarity of induced effects could mean a similar way of their action. At present, for the case of plants it looks clear enough and is, probably, realized both via direct action on cell membranes and via specific gene expression fol-

lowed by the initiation of the corresponding secondary processes. The data mentioned above for BS properties in insects could be interpreted as an indication on the same mechanism of action for them, at least at the genetic level. Although the high structural similarity of BS and ES could be a reason of the same gene expression under the action of each hormone, the existence of genes specifically initiated by BS in insects cannot be excluded. If the last is true also for higher animals, it gives an easy explanation of all the observed phenomena. In such a case, also similar types of the receptors could be involved in signal transduction that makes actual a search for them in animals and other organisms outside plant kingdom. One of the explanations of non-specificity of BS-action could be their involvement in bioregulation at a very low downstream control point where most of vital signalling pathways of higher levels can be greatly influenced. In plants, for example, one of such control points could be the beginning of the light-signalling sequence between the photoreceptors and initiation of other phytohormones [167,168], which start playing their roles at a later stage and in a more specialized manner. A possible key for an intriguing problem: why the BS action is often realized in adaptogenic effects (including all kinds of protective properties, such as toxyco-protection, radio-protection, stress-resistance increase, etc.) together with growth stimulation, might be the recent finding in plants of the close similarity of genes involved in BS signal transduction with genes responsible for some protective properties, such as disease-resistance [169] for example. Nowadays, more and more data show a tremendous role of hydroxylated sterols in human bioregulation [170]. Recognizing the discussed properties of BS, which are typical representatives of hydroxylated sterols, make them promising leads for the discovery of new pharmacological agents and new approaches to medicinal treatment of diseases. Finding the discussed stimulating-protective activities of BS in many organisms and wide natural consumption of BS by all phytophagous animals allows proposing their essential role as food components, a kind of vitamins, involved in bioregulation at the most basic level such as adaptation to the environment including protection against the stresses of different origin. These properties as well as medical prospects of BS, which are clearly designated now, strongly support our idea expressed fifteen years ago [4] that the data on BS-action in non-plant organisms ‘‘promise further findings that may become important for humans’’. This idea is still relevant for researchers working in the area. Acknowledgments The authors are indebted to the Belarusian Foundation for Fundamental Research for financial support (Projects X13K-094, X13Mld-009, and X14P-139). References [1] Fieser LF, Fieser M. Steroids. New York: Reinhold Publishing Corporation; 1959. [2] Akhrem AA, Levina IS, Titov YA. Ecdysones - Steroidal hormones of insects. Minsk: Science and Technique; 1973. [3] McMorris TC. Antheridiol and oogoniol, steroid hormones which control sexual reproduction in Achlya. Phil Trans Roy Soc Series B 1978;248:459–70. [4] Khripach VA, Zhabinskii VN, de Groot A. Brassinosteroids. A new class of plant hormones. San Diego: Academic Press; 1999. [5] Khripach V, Zhabinskii V, de Groot A. Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI century. Ann Bot 2000;86:441–7. [6] Ikekawa N, Zhao YJ. Application of 24-epibrassinolide in agriculture. In: Culter HG, Yokota T, Adam G, editors. Brassinosteroids. Chemistry, Bioactivity and Applications ACS Symposium Series; 1991. p. 280–91. [7] Kuzmitskii BB, Mizulo NA. Technical report ‘‘study of acute toxicity of epibrassinolide and its preparative forms’’. Minsk: Institute of Bioorganic Chemistry, Academy of Sciences of Belarus; 1991.

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