Biotic and abiotic elicitors induce biosynthesis and accumulation of

Romanian Biotechnological Letters Vol. 16, No. 6, 2011 Copyright © 2011 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

6683

Biotic and abiotic elicitors induce biosynthesis and accumulation of resveratrol with antitumoral activity in the long - term Vitis vinifera L. callus cultures

Received for publication, May 8, 2011

Accepted, June 20, 2011

RALUCA MIHAI¹*, STURZOIU CRISTINA², FLORENTA HELEPCIUC¹, AURELIA BREZEANU¹, GHEORGHE STOIAN² *corresponding author: Dep. Plant and Animal Cytobiology, Institute of Biology , Bucharest -Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, Phone + 4 021 2219202, Fax + 4 021 2219071; e-mail: [email protected] ¹ Institute of Biology , Bucharest - Romanian Academy ² Research Center of Biochemistry and Molecular Biology, University of Bucharest

Abstract

The present study investigated the effect of some biotic (fungal extract of Fusarium oxysporum) and abiotic elicitors (mannitol, abscisic acid, jasmonic acid) at different concentrations and combined treatments on the accumulation of resveratrol in a long-term Vitis vinifera callus culture. The application of a two- stage culture system with a combined treatment of mannitol (2mM) and jasmonic acid (40µM) resulted in the optimum accumulation of resveratrol in the callus biomass. Some preliminary investigations on the antitumoral properties of the treated callus extracts on a hepatocellular carcinoma HepG2 cell lines revealed that a high activity was achieved by the extracts from the treated callus with the combined elicitors mannitol (2mM) and jasmonic acid (40µM). The obtained data are sustained by the high accumulation of resveratrol in the callus biomass. This stilbene-type compound is recognized as a remarkable compound with diverse biological effects, having tremendous potential as a chemopreventive and/or chemotherapeutic agent in clinical medicine.

Keywords: biotic elicitors, abiotic elicitors, resveratrol, antitumoral activity Introduction

Plant secondary products are of immense use as potential drugs, nutraceuticals and food additives. Some molecules belonging to this class have been identified because of their roles as protective agents. Special attention has been given to trans-resveratrol (trans-3,4`,5-trihydroxystilbene) due to its proved relation with anti-initiation, anti-promotion and anti-progression activities in malignant tumors [1] and its recognition as chemotherapeutic agent in humans, acting upon leukaemia and breast carcinoma cells [2].

Due to the limited availability and complexity for chemical synthesis, plant cell culture becomes an alternative route for large-scale production of this desired compound [3]. The applied process of elicitation, that makes use of the plant capacity and plant cell cultivated in vitro to react to various stress stimuli by a number of protective reactions, lead to an increase accumulation of the secondary metabolite. Trans – resveratrol, a phytoalexin of a phenolic nature, belonging to the stilbene family is one of antifungal compounds in grapes [4]. Biotic elicitors and abiotic stresses stimulate its in vivo and in vitro synthesis. Vitis vinifera cell cultures have been used in several studies to explore the factors involved in the induction and regulation of stilbene biosynthesis and metabolism [5].

The aim of this study was to evaluate in a two-stage culture system the elicitor effect of some biotic (fungal extract of Fusarium oxysporum) and abiotic factors (mannitol, jasmonic acid and abscisic acid) on Vitis vinifera L. long-term callus culture regarding the

RALUCA MIHAI, STURZOIU CRISTINA, FLORENTA HELEPCIUC, AURELIA BREZEANU, GHEORGHE STOIAN

6684 Romanian Biotechnological Letters, Vol. 16, No. 6, 2011

resveratrol biosynthesis and accumulation along with a preliminary investigation on the antitumoral effects of the callus extracts. This research was achieved by carrying out a quantitative analysis of their role in resveratrol production. The antitumoral activities of Vitis vinifera L callus extracts in response to elicitor culture system was achieved by a MTT test for revealing the cellular viability of a hepatocellular carcinoma Hep G2 cell line under the influence of the tested callus extracts.

Materials and methods Plant Material and Extraction Procedures

The study was conducted using a long-term Vitis vinifera L. cv. Isabelle pericarp callus, maintained through periodic subcultivations on a variant of basal Gamborg-B5 (1968) medium, supplemented with 0.1 mg/l NAA (α-naphtalene acetic acid), 0.2 mg/l kinetine, 2 g/l casein hydrolysate, 30g/l sucrose, 8g/l agar (Difco), which has been referred in the experiment as the control variant. To investigate the influence of different types and concentrations of elicitors as regards the resveratrol accumulation in the callus cells, a two-stage experiment was performed. Table 1. Types of elicitors and the concentrations used in the two-stage culture system.

Seven variants of the experimental medium were analyzed (Table 1) for 37 days and

each treatment was repeated 3 times. For the first stage of the experiment the long- term callus was grown 30 days on the medium variants supplemented with mannitol in concentration 2mM to ensure the callus proliferation. Further subcultivation of the callus for 7 days during the second stage of the experiment ensured the biosynthetic capacity of the callus. The fungi extract was obtained by centrifugation of Fusarium oxysporum sonicated pellets culture and the fungi medium was represented by the supernatant filtrate. All the abiotic elicitors used, with the exception of mannitol, were dissolved in 1ml MeOH. For accurate research results the influence of MeOH on the culture was also tested, named as Blank experimental medium variant. Sample preparation and extraction of trans-resveratrol

A solvent extraction system assisted by ultrasonication was introduced to run at room temperature according to the method of Romero-Perez et. al., 2001 [6], with slightly modifications. A methanol/0.1% HCl 80:20 (v/v) solutions was used as a solvent in the study. The ratio of callus sample weight to solvent volume was 1:1, so three samples, each one of 3 g callus from the all medium variants, were extracted with 3ml solvent solution. The frequency of the ultrasonic processor was 47 kHz. The extract obtained by ultrasonication-assisted method was kept overnight at 40 C in refrigerator for 12 hours. The insoluble material


Romanian Biotechnological Letters, Vol. 16, No. 6, 2011 6685

was removed by centrifugation (4000 rpm, 15 min) and the extracts were concentrated by evaporation and used for further analysis. Calibration and determination of trans-resveratrol concentration using the HPLC analysis

The resveratrol compound used for further HPLC analysis was dissolved in methanol at a concentration of 1 mgl-1 and stored away from direct light at 4 0C until used. The standard methanolic solution of resveratrol was diluted in a range from 10µgl-1 to 10mgl-1 to create a calibration curve necessary for further resveratrol quantification in Vitis calli. The Trans-resveratrol content in the analyzed callus extracts was determined by the HPLC method using the JASCO Model AS-2051 liquid chromatograph equipped with Jasco MD-2015 Plusmultiwavelenght Detector and the Jasco FP-2020 Plus Inteligent Fluorescent Detector coupled to the PROGRAM Jasco-Chrompass Chromatography Data System. The sample was injected into HPLC system, using a 100 C18 5 UM 25X 0, 4 reversephase Nucleosil column. The separation was carried out at room temperature. The isocratic elution at a flow rate of 1.0 ml/min used the mobile phase A of 1% acetic acid in demineralized water, and the mobile phase B of 100% methanol. For the detection of trans- resveratrol, the optimal wavelength of 312 ± 2 nm was used. Measurement of cell viability and proliferation

The hepatocellular cells were incubated at 37°C, 5% CO2 in the presence of the treatment with resveratrol contained by the tested probes of calli, at different times. After 24 hours and 48 hours the cells were submitted to the viability MTT test (3-(4,5-dimethylthiazolium-2-yl)-2,5-diphenyltetrazol bromide). For this reason, at the tested times, the treatment was removed and the cells washed from the PBS solution and then incubated for 2 hours at 37 °C with MTT solution. The formazan crystals formed in the viable cells were dissolved in 250 µl isopropanol/wells and transferred in culture plates with 96 wells. The absorbance was measured at 595 nm using a spectrophotometer UV-VIS (Jasco V-650, Japan). The proliferation rate was calculated by comparison with the control culture (cells cultured on plastic), considered 100% (proliferation rate).There was also tested a probe with NaHCO3

for revealing the influence of this compound on the HepG2 cell line, that was further used (200 µL NaHCO3 3%) for adjusting the pH at 7-8 for all the analyzed probes. Cellular morphology

The morphology of the hepatic tumoral cells was evaluated by optic microscopy analyses. The human cells were cultivated on culture dishes with 24 wells and incubated at 370C, till the confluence for 24 hours, and then the tested substances were added to the cellular culture.

The cultivated HepG2 cell line for 48 hours, (the control and the others grown in the presence of the tested callus extracts) were photographed using a contrast phase microscope Olympus before washing and incubation with MTT for 2 hours. The determinations were performed in comparison with the human hepatic tumoral cells cultivated in the absence of the tested substances. Results and Discussion

1. Trans-resveratrol concentrations at callus culture level Under a pathogen attack, plants evolve sophisticated systems of detection and response to decipher the pathogen signals and to induce appropriate defenses. These systems include specific networks that operate through the action of signaling molecules such as jasmonate and generate the accumulation of pathogenicity-related proteins, phytoalexins [7].



020406080

100120140

Control

Blank

Man- Fungi m

edium

Man- ABA 20

Man-Man20

Man- Fungi extra

ct

Man- JA 40

Medium variants

Res

vera

trol c

once

ntra

tion

(µg/

mL)

Trans-Resveratrol (3,5,4′-trihydroxystilbene) is the major component of the phytoalexin response of the plant. Jasmonic acid has been proposed as key compound of the signal transduction pathway involved in the elicitation of secondary metabolite biosynthesis which takes part in plant defense reactions [8]. Application of jasmonic acid on grapevine leaves and plant cell suspension cultures can induce the accumulation of stilbenes (resveratrol) [9]. This could be an explanation for the highest reported amount of trans-resveratrol in the callus cells

cultured in the two-stage system involving the presence of mannitol (2mM) and jasmonic acid (40µM).(Fig.1) Fig. 1. Resveratrol content of the callus extracts grown under the influence of the tested abiotic and biotic elicitors in a two-stage system. Biotic and abiotic elicitors have different mechanisms of elicitation and, when used in combination, synergistically enhance metabolite production in cultured cells [10]. This is also the case of resveratrol accumulation in Vitis vinifera L. callus culture under the influence of biotic and abiotic elicitor combinations, such as fungal extract of Fusarium oxysporum and mannitol. It is known that the positive influence of the fungal extract on resveratrol accumulation can be explained by the fact that this metabolite is accumulated in grape skin in response to various fungal infections [11].

2. HepG2 cell viability and proliferation The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell viability. The yellow tetrazolium MTT (3-(4, 5- dimethylthiazolyl-2)-2, 5 diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. So, the variation of viability of cultivated HepG2 cell line in the presence of the tested callus extracts cultivated in a two-stage system under the influence of different combination of elicitors, was measured spectrophotometrically.

The tested extracts proved to inhibit the cellular viability and proliferation of the human hepatocellular cell line, the highest positively effects being achieved by the extracts treated with the elicitors mannitol and jasmonic acid (40µM), followed by that under the influence of two-stage culture system with Fusarium oxysporum fungal extract (1ml) and mannitol (1 mM) both after 24 hours of treatment (Fig. 1) and 48 hours (Fig. 2).



Fig.1. The viability of the HepG2 cell line after 24 hours treatment in the presence of callus extracts grown in a two-stage culture system with different elicitor combinations.

The data illustrated that the inhibition of the HepG2 cell line proliferation by the callus extracts cultivated in these two-stage systems was due to the high amount of trans-resveratrol present at the level of this callus extracts. It is known that trans-resveratrol exhibits antitumoral properties, as suggested by its ability to suppress proliferation of a wide variety of tumor cells, including lymphoid and myeloid cancers, multiple myeloma, cancers of the breast, prostate, stomach, colon, pancreas and thyroid, melanoma, head and neck squamous cell carcinoma, ovarian carcinoma and cervical carcinoma [12].

Fig.2. The viability of the HepG2 cell line after 48 hours treatment in the presence of callus extracts grown in a two-stage culture system with different elicitor combinations. Cellular morphology

Under microscopic examination, the HepG2 cells presented a normal phenotype after growing for 24 and 48 hours in the absence of callus extracts, representing the control system (Fig. 3, 4).

CONTROL MARTOR(NaHCO3)

Man-JA40

Man-ABA20

Man-Fungi extract

Man-Fungi medium

0

0,2

0,4

0,6

0,8

1

1,2

SAMPLES

DO 595nm

CONTROL MARTOR (NaHCO3)

Man-JA40

Man-ABA20

Man-Fungi extract

Man-Fungi medium

00,1

0,20,3

0,40,5

0,60,7

0,80,9

1

SAMPLES

DO 595nm



Fig. 3. Hep G2 cellular proliferation after 24 h of incubation in the absence of the callus extract

Fig. 4. Hep G2 cellular proliferation after 48 h of incubation on culture medium in the absence of the callus extracts.

After 24 and 48 hours of incubation, the cells cultivated in the presence of the tested

callus extracts under the influence of combined elicitors mannitol (1mM) and jasmonic acid (40µM), did not maintained the phenotype characteristics of normal HepG2 cell line (Fig. 5, 6), demonstrating an inhibition of cell viability and proliferation.

Fig. 5. The phenotype characteristics of HepG2 cells after 24h cultivation in the presence of the tested callus extracts under the influence of combined mannitol (1mM) and jasmonic acid (40 µM)

Fig. 6. The phenotype characteristics of HepG2 cells after 48h cultivation in the presence of the tested callus extracts under the influence of combined elicitors- mannitol (1mM) and jasmonic acid (40µM).

Conclusions

This study demonstrated that the two-stage culture system with combined treatment of elicitors - mannitol (1mM) along with jasmonic acid (40µM) - enhanced significantly the trans-resveratrol biosynthesis in the callus grown under these conditions, as demonstrated by HPLC determinations. The MTT achieved test proved that extract of this callus inhibits strongly the HepG2 cell line proliferation and viability, due to the presence of a high amount of trans-resveratrol, known as a strong anti-cancerous agent. The microscopy images showed that the tested samples of the callus extracts grown in response to mannitol and jasmonic acid influence, induce morphological modifications in the cultured human hepatocellular cell line compared to the control. Acknowledgments

The research was partially supported by the Institute of Biology, Bucharest - Romanian Academy, as integral part of the PhD thesis achievement.



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Biotic and abiotic elicitors induce biosynthesis and accumulation of