Taxol, an anticancer drug produced by an endophytic fungus ...

World J Microbiol Biotechnol (2008) 24:717–724 DOI 10.1007/s11274-007-9530-4

ORIGINAL PAPER

Taxol, an anticancer drug produced by an endophytic fungus Bartalinia robillardoides Tassi, isolated from a medicinal plant, Aegle marmelos Correa ex Roxb. V. Gangadevi Æ J. Muthumary

Received: 21 March 2007 / Accepted: 5 August 2007 / Published online: 19 August 2007 Springer Science+Business Media B.V. 2007

Abstract Taxol is an important anticancer drug widely used in the clinic. An endophytic fungus Bartalinia robillardoides (strain AMB-9) was isolated from Aegle marmelos, a medicinal plant and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed chromatographically and spectrometrically, for the presence of Taxol. The amount of taxol produced by this endophytic fungus was quantified by HPLC. It produced 187.6 lg/L of taxol which suggests that the fungus can serve as a potential material for genetic engineering to improve the production of Taxol. This fungal taxol isolated from the organic extract of this fungal culture, has strong cytotoxic activity towards BT 220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro, tested by Apoptotic assay. Keywords Fungal endophyte Medicinal plant Taxol Anticancer drug

Introduction Taxol is a complicated diterpenoid compound with anticancer properties, which was first isolated from Taxus brevifolia (Wani et al. 1971). Its mode of action is unique as it prevents the depolymerization of tubulin during the

V. Gangadevi J. Muthumary (&) Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600 025, India e-mail: [email protected] V. Gangadevi e-mail: [email protected]

processes of cell division (Schiff et al. 1979). The most common source of taxol is the bark of trees belonging to the Taxus family including Yew trees. Unfortunately, these trees are rare, slow growing, and a large amount of bark may have to be processed to obtain a small amount of the drug. The amount of taxol found in yews is relatively small, ca 0.01–0.03% dry weight and this has been a major factor in contributing to its high price in market. With the discovery that certain endophytic fungi are able to produce taxol has lead to the possibility of a cheaper and more widely available product which may eventually be available via industrial fermentation (Stierle et al. 1993; Strobel et al. 1996). Over the last decade there has been a great deal of interest in finding out other fungi, which produce taxol. The discovery of Taxomyces andreanae, was the first report that any organism other than Taxus spp. can produce taxol (Stierle et al. 1994). However, the yields of taxol and taxanes have been low. Taxol has been reported in a novel endophytic fungus T. andreanae but also has been demonstrated to occur in a number of unrelated fungal endophytes (Strobel 2003). Endophytic fungi are being explored by both pharmaceutical and agricultural industries as they represent an untapped pool of secondary metabolites (Petrini et al. 1992; Dreyfuss and Chapela 1994). In recent years, the quest for the isolation of new compounds from medicinal plants has become a fascinating area of research. The purpose of this work is to identify a taxolproducing endophytic fungus from the selected medicinal plant and study its anticancer activities. In the present study, Bartalinia robillardoides, a coelomycetous fungus, isolated as endophyte from the leaves of Aegle marmelos Correa ex Roxb. (Rutaceae) was shown to produce the drug. Virtually very few reports are available on the association of endophytic fungi from tropical medicinal plant species. Therefore, this study provides first report on

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taxol production by fungal endophyte of medicinal plant from Southern India.

Materials and methods Isolation and identification of endophytic fungi The fungus used in this study is one of the twenty endophytic fungi isolated from the leaves of medicinal plants in Chennai city, India. The leaf samples were surface sterilized by the modified method of Dobranic et al. (1995). The leaves were thoroughly washed in running tap water and small pieces of approximately 0.5 cm diameter were cut with the aid of a flame-sterilized cork borer. Then the leaf discs were surface sterilized by immersion in 70% ethanol for 5 s, followed by 4% sodium hypochlorite for 90 s and then rinsed in sterile distilled water for 10 s. The excess moisture was blotted in a sterile filter paper. The surface sterilized leaf segments were evenly spaced in petri dishes (9 cm diameter) containing potato dextrose agar (PDA) medium (amended with chloramphenicol 150 mgL–1). The petri dishes were sealed using ParafilmTM and incubated at 26 ± 1 C in a light chamber with 12 h of light followed by 12 h of dark cycles. The petri dishes were monitored every day to check the growth of endophytic fungal colonies from the leaf segments. The hyphal tips, which grew out from leaf segments were isolated and subcultured onto PDA and brought into pure culture. The isolated endophytic fungi were identified using standard monographs. The identified cultures of endophytic fungi were deposited at the Madras University Botany Laboratory (MUBL), CAS in Botany, University of Madras, Chennai—600 025. The immediate concern is to find one or more fungi that produce more taxol. An endophytic fungus B. robillardoides strain AMB-9 (MUBL No. 662), was screened for taxol production. Photomicrographs of conidia were taken with the help of Carl Zeiss Axiostar plus-Photomicroscope (phase contrast) with Nikon FM 10 Camera and Nikon HFX Labophot (bright field) with Nicon FX-35A by using Konica films.

Preparation of fungal extracts The endophytic fungus was grown in 2 L Erlenmeyer flasks containing 500 mL of MID medium supplemented with soytone (Pinkerton and Strobel 1976) and incubated for 21 days. After 3 weeks of still culture at 26 C, the culture fluid was passed through four layers of cheese cloth to remove solids and extracted with organic solvent. The extraction and isolation procedure followed was that of Strobel et al. (1996). After methylene chloride extraction,

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Fig. 1 (A) Petriplate showing the growth of the fungus Bartalinia robillardoides (B)Conidia: 400 X

the organic phase was collected and the solvent was then removed by evaporation under reduced pressure at 35 C using rotary vacuum evaporator. The dry solid residue was re-dissolved in methanol for the subsequent separation and extracts were analyzed by chromatographic separation and spectroscopic analyses. The standard taxol (Paclitaxel) was purchased from SIGMA.

Chromatographic separation All comparative Thin Layer Chromatographic analyses were carried out on Merck 0.25 mm silica gel plates, developed in the following solvents: (a) chloroform/


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Fig. 2 High performance liquid chromatogram of standard taxol (A) and fungal taxol (B); the mobile phase was methanol/ acetonitrile/water (25:35:40, by vol.), flow rate at 1.0 mL/min; Retention time of standard taxol: 10.69 min; retention time of fungal taxol: 10.56

methanol (7:1 v/v); (b) chloroform/acetonitrile (7:3 v/v); (c) ethylacetate/2-propanol (95:5 v/v); (d) methylene chloride/tetrahydrofuran (6:2 v/v); (e) methylene chloride/ methanol/dimethylformamide (90:9:1v/v/v). The presence of taxol was detected with 1% w/v vanillin/sulphuric acid reagent after gentle heating (Cardellina 1991). Appearance of a bluish spot of fading to dark gray after 24 h indicates the presence of taxol. To further confirm the presence of taxol, the fungal sample was subjected to high performance liquid chromatography. Taxol was analyzed by HPLC (Shimatzu 9A model) using a reverse phase C18 column with a UV detector. Twenty microlitres of the sample was injected each time and detected at 232 nm. The mobile phase was methanol/acetonitrile/water (25:35:40, by vol.)

at 1.0 mL min–1. The sample and the mobile phase were filtered through 0.2 lm PVDF filter before entering the column. Taxol was quantified by comparing the peak area of the samples with that of the standard taxol.

Spectroscopic analyses The purified sample of taxol was analyzed by UV absorption, dissolved in 100% methanol at 273 nm in a Beckman DU-40 Spectrophotometer and compared with standard taxol. 1H NMR spectrum was recorded to confirm the structure of fungal taxol at 23 C in CDCl3 using a JEOL GSX 500 spectrometer (operating at

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Fig. 3 UV absorption spectrum of standard taxol (A) and fungal taxol (B) was recorded in methanol at 273 nm

499.65 MHz) and were assigned by comparison of chemical shifts and coupling constants with those of related compounds. Chemical shifts were reported as dvalues relative to tetramethylsilane (TMS) as internal reference, and coupling constants were reported in Hertz.

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The FAB mass spectra were recorded on a JEOL SX 102/ DA-6000 Mass Spectrometer/Data System using Argon/ Xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was 10 kV and the spectra were recorded at room temperature.


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Fig. 4 Fast atom bombardment mass spectrum of standard taxol (A) and fungal taxol (B). The accelerating voltage was 10 kV and recorded at room temperature

Cytotoxicity of fungal taxol by apoptosis Cytotoxicity effect of fungal taxol isolated from the endophytic fungus, B. robillardoides, was detected and quantified using apoptotic assay by the method of Ruckdeschel et al. (1997) on various cancer cells, at different concentration. All the cell lines used in this study were obtained from National Centre for Cell Sciences (NCCS), Pune. The morphological changes of the cancer cells which were treated with different concentrations of taxol ranging from 0.005 lM to 5 lM were studied at different times during incubation period for 24, 48 and 72 h. The cell

DNA was stained with 0.5 mg/mL propidium iodide in PBS for 15 min and destained in PBS. After treatment with different concentrations of fungal taxol, the cell morphology was determined by light microcopy. In all, five different fields were randomly selected for counting at least 500 cells. The percentage of apoptotic cells was calculated for each experiment. Cells designated as apoptotic were those that displayed the characteristic morphological features of apoptosis, including cell volume shrinkage, chromatin condensation and the presence of membranebound apoptotic bodies. For each experiment, 500 cells were counted. The % of apoptotic cells in apoptosis were

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Fig. 5 1H NMR spectrum of standard taxol (A) and fungal taxol (B) in CDCl3 at 500 MHz. The chemical shifts in ppm high frequency from TMS. The structure of taxol is shown as an insert

calculated by dividing the number of apoptotic cells by total number of cells · 100.

Results and discussion The anticancer drugs, which are available at present, possess enormous side effects and are not effective against many forms of cancer. Taxol has a unique mode of action when compared with other anti-cancer compounds. The aim of this present study, is to isolate and identify the taxol-producing endophytic fungi from medicinal plants, so that the fungus can serve as a potential material for fungus engineering to improve the production of Taxol. Based on the morphology of the mycelial colony as well as the characteristics of the conidia, the endophytic fungus was identified as Bartalinia robillardoides (Fig. 1) and screened for its taxol production. The extract of fungal culture was examined for the presence of taxol by chromatographic and spectroscopic analyses. Taxol, produced by the fungus was detected using a spray reagent consisting

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of 1% vanillin (w/v) in sulfuric acid after gentle heating. It appeared as a bluish spot fading to dark gray after 24 h. The compound has chromatographic properties identical to standard taxol in solvent systems A-E, and gives colour reaction with the spray reagent and they had Rf values identical to that of standard taxol. Therefore, it was evident that this fungus showed positive results for taxol production. In HPLC analysis, the fungal extract gave a peak when eluting from a reverse phase C18 column, with a similar retention time as standard taxol (Fig. 2). The amount of taxol produced by this fungus in liquid culture was 187.6 lg/L. The biggest problem of using fungi in fermentation was less production of taxol, its very low yield, and unstable production. The taxol yield of such reported fungi varies from 24 ng to 70 ng per litre culture (Stierle et al. 1993; Strobel et al. 1996). Although the amount of Taxol produced by most of the endophytic fungi associated with taxus trees is relatively small when compared with that of the trees, the short generation time and high growth rate of fungi make it worth while to continue our investigation of these species.


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Table 1 Taxol-induced apoptosis by the endophytic fungus B. robillardoides in various cancer cell lines S. No.

Cell lines

1.

BT 220 (Breast)

2.

H 116 (Human colon)

Taxol content (lM) 0

Int 407 (Human intestine)

15.8

0.05 0.5

75.5 80.6

5

21.3

0

5.

HL 251 (Human lung)

HLK 210 (Human leukemia)

0 13.6

0.05

65.8

0.5

76.3

5

36.3

0 0.005

4.

0

0.005

0.005

3.

Apoptotic cell (%)

0 15.8

0.05

68.6

0.5

79.5

5

18.8

0

0

0.005

12.8

0.05 0.5

69.3 71.8

5

32.9

0 0.005

0 21.6

0.05

66.5

0.5

76.3

5

30.8

The presence of taxol in the fungal extract was further confirmed by UV spectroscopy. The UV spectral analysis of the fungal taxol is given in Fig. 3 and the spectrum was superimposed on that of standard taxol at 273 nm. Further convincing spectroscopic evidence for the identity of taxol was confirmed by FAB mass spectroscopy. The FAB mass spectrum of fungal taxol isolated from B. robillardoides is given in Fig. 4. It was evident that the diterpenoid taxol was much more complex since its molecular weight from high-resolution mass spectrometry is C47H51NO14, corresponding to a molecular weight of 853. Characteristically, standard taxol yielded MH+ at m/z 854. By comparison, fungal taxol also yielded a peak MH+ at m/z 854 with characteristic fragment peaks at 569, 551, 509, 464, 286 and 268. Major fragment ions observed in the mass spectrum of taxol can be placed into three categories which represents major portions of the molecule (McClure and Schram 1992). The peaks corresponding to taxol, exhibited mass-to-charge (m/z) ratios corresponding to the molecular ions (M+H)+ of standard taxol (854) confirming the presence of taxol in the fungal extracts. As reported in detail by

Wani et al. (1971), the esterified position was found to be the allylic C13 hydroxyl moiety. The FAB mass spectrum of the fungal sample not only corroborated the molecular formula, C47H51NO14, but was found to be identical with FAB-MS of standard taxol. In 1H NMR spectra, almost all of the signals are well resolved and are distributed in the region from 1.0 ppm to 8.5 ppm. The strong three-proton signals caused by the methyl and acetate groups lie in the region between 1.0 ppm and 2.5 ppm, together with multiplets caused by certain methylene groups. Most of the protons in the taxane skeleton and the side-chain are observed in the region between 2.5 ppm and 7.0 ppm, and the aromatic proton signals caused by the C-2 benzoate, C30 phenyl and C-30 benzamide groups appear between 7.0 ppm and 8.3 ppm. The characteristic chemical shifts of taxol are shown in Fig. 5. The taxol assignments obtained was confirmed with the earlier reports (Chmurny et al. 1992). Cytotoxicity effect of fungal taxol from B. robillardoides was further tested using apoptotic assay on various cancer cells viz., human breast cell BT220, human colon H116, human intestine Int407, human lung HL251 and human leukemia HLK 210. It is indicated that with the increase of taxol concentration from 0.005 lM to 0.05 lM, taxol induced increased cell death through apoptosis. With further increase of taxol concentration from 0.05 lM to 0.5 lM, the taxol-induced cell death through apoptosis only increased slightly. When the taxol concentration was increased from 0.5 lM to 5 lM, the taxol-induced cell death through apoptosis decreased dramatically (Table 1). In the present study, it was observed at low to medium concentration (0.005–5 lM), the efficacy of fungal taxol was quite dependent on the specific cell type. This is in agreement with the results of Yeung et al. (1999). It has been reported that taxol at low concentrations (nM) induces cell apoptosis and the efficacy of taxol is quite dependent on the specific cell type. This also supports the previous findings of other groups that at low concentration, taxol inhibits cell proliferation by blocking mitosis. Acknowledgements We thank Dr. N. Anand, Director, CAS in Botany, University of Madras for the laboratory facilities provided. One of the author (Gangadevi) is thankful to the Ministry of Environment and Forests, Government of India (Project No.14–53/2001ERS-RE dt. 9.12.2003) for the Junior Research Fellowship during which the investigation was carried out.

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724 Dobranic JK, Johnson JA, Alikhan QR (1995) Isolation of endophytic fungi from eastern larch (Larix laricina) leaves from New Brunswick, Canada. Can J Microbiol 41:194–198 Dreyfuss MM, Chapela IH (1994) Potential of fungi in the discovery of novel, low-molecular weight pharmaceuticals. In: Gullo VP (ed) The discovery of natural products with therapeutic potential. Butterworth-Heinemann, London, pp 49–80 McClure TD, Schram KH (1992) The mass spectrometry of taxol. J Am Soc Mass Spectrom 3:672–679 Petrini O, Sieber TN, Toti L, Viret O (1992) Ecology, metabolite production and substrate utilization in endophytic fungi. Nat Toxins 1:185–196 Pinkerton R, Strobel G (1976) Serinol as an activator of toxin production in attenuated cultures of H. sacchari. Proc Nat Acad Sci USA 73:4007–4011 Ruckdeschel K, Roggenkamp A, Lafont V, Mangeat P, Hessmann J, Rouot B (1997) Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis. Infect Immun 65:4813–4821 Schiff PB, Fant J, Horowitz SB (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277:665–667

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