DrugRes/2014-12-0933/21.5.2015/MPS
Original Article
Cytotoxic Potential and Molecular Characterization of Fungal Endophytes from Selected High Value Medicinal Plants of the Kashmir Valley – India
Authors
R. A. Dar1, P. H. Qazi1, I. Saba1, S. A. Rather1, Z. A. Wani2, A. K. Qazi2, A. A. Shiekh1, A. Manzoor1, A. Hamid2, D. M. Modae2
Affiliations
1
Key words ▶ ehrlich ascites carcinoma ● ▶ SRB ● ▶ secondary metabolites ●
Abstract
Biotechnology Division, CSIR – Indian Institute of Integrative Medicine, Sanatnagar – Srinagar, Kashmir, India Cancer Pharmacology Division, CSIR – Indian Institute of Integrative Medicine, Jammu – Tawi, India
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The present study explores the fungal endophytes from selected high value medicinal plants to check their activities at in-vitro and in-vivo level. The in-vitro cytotoxicity of selected endophytes revealed potent growth inhibition against human cancer cell lines of leukemia (THP-1), lung (A549), prostate (PC-3), colon (Caco-2), neuroblastoma (IMR-32) and breast (MCF-7) at a concentration of 100 µg/ml. Among them the endophytic strains i. e., IIIM2, IIIM3, IIIM7 and IIIM8 showed most significant growth inhibition
Introduction
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received 28.12.2014 accepted 05.05.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1550042 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence P. H. Qazi Biotechnology Division Indian Institute of Integrative Medicine (CSIR) Sanatnagar – Srinagar 190005 Kashmir India Tel.: + 911/94/2431 255 Fax: + 911/94/2430 779 [email protected]
In the past few decades, scientists have realized that plants act as a reservoir of unexplored potential beneficial microorganisms known as endophytes. These endophytic microbes can produce secondary metabolites such as enzymes, growth hormones, antimicrobial, antifungal or anticancer substances [1, 2]. The discovery of taxol producing endophytic fungus (Taxomyces andreanae) derived from Pacific yew (Taxus brevifolia) was unexpected [3]. Fungal endophytes living in association with medicinal plants have received increasing attention after the discovery that the endophytic fungus Taxomyces andreanae and Tubercularia spp., recovered from Taxus brevifolia and Taxus mairei respectively, also produce the anticancer drug paclitaxel (Taxol) [4, 5]. The discovery that rare, valuable plant products might also be produced by their endophytic microorganisms is of special pharmacological interest [6]. Various workers around the globe have demonstrated that the endophytic fungal communities living within medicinal plant tissues produce a wide range of metabolites with different biological activities [7, 8, 9] that may be
against colon (Caco-2), prostate (PC-3), lung (A549) and leukemia (THP-1) cancer cell lines. At the in-vivo level maximum (58.95 %) tumor growth inhibition was documented with the extract of IIIM2 against Ehrlich Ascites Carcinoma mouse modal. All the potent fungal endophytic strains were characterized using ITS 4 and ITS 5 region sequencing and phylogenetic analysis was ascertained among them. This paper confirms the 2 elite endophytic fungal strains, IIIM2 and IIIM8, have the potential to act as a source of new anticancer compounds.
useful as scaffolds for the development of new drugs. As per reports a wide variety of natural products can be obtained from endophytic microbes [10, 11]. Endophytes associated with the plants producing bioactive natural products also produce the same natural products due to host-endophyte co-evolution [12]. Endophytes have been intensively studied in several unexplored environments around the world. There is a huge interest across the world to determine the biodiversity of endophytic mycoflora within the plant kingdom with the hope of discovering unique endophytes that produce novel compounds that are beneficial to man [13]. To harness the potential of hidden treasure, endophytes have been isolated from indigenous plant species collected from the Kashmir valley in the Indian state of Jammu & Kashmir. In the present study an attempt was made to isolate endophytic fungi from selected high value medicinal plants of Kashmir valley and assessed their in-vitro and in-vivo antitumor potentials. The aim of the study was to assess potential of the endophytic fungi as sources of anticancer compounds which can be used as drugs in the treatment of cancer.
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
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Materials and Methods
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Collection and identification of plant material
The selected high value medicinal plants were collected from 2 regions, Sonamarg, Jammu and Kashmir, India (Latitude: 34 °18’17.00 N, Longitude: 75 °17’08.00 E, Altitude: 8711 feet) and the field station of CSIR-Indian Institute of Integrative Medicine, Sanantnagar, Kashmir – India (Latitude: 34 °01’54.29 N, Longitude: 74 °47’53.12 E, Altitude: 5220 feet). 4 types of plants in 5 replicates were collected randomly from each region by individual and group visits (Rheum emodi wall. ex Meissn and Hypercum perforatum L. from Sonamarg and Diocoria deltodia Wall. and Artemisia annua L. from the field station of IIIM Sanantnagar). A voucher specimen of each plant was submitted to the Division of Plant Systematic and Taxonomy, CSIR-IIIM – Jammu for identification and authentication.
Isolation, purification and extraction of different endophytic strains
The endophytic fungal strains were isolated, purified and preserved in glycerol [14]. For extraction, the endophytic fungal strains were grown on potato dextrose broth at 180 rpm, 28 °C for 10–15 days in an incubator shaker (New Brunswick, USA). The grown culture of each endophyte was homogenized with 10 % methanol under ultra sonicator. The fermented broth was then extracted with dichloromethane 3 times after which Na2SO4 (40 μg/ml, Merck) was added to further remove the aqueous layer within the mixture. Further the mixture was transferred to a round bottom flask and dried using a rotary evaporator and weighed to constitute the crude broth extract.
In-vitro cytotoxicity assay
The in-vitro cell cytotoxicity was checked by sulphorhodamine B (SRB) assay. In this assay cell suspension of optimum cell density was seeded and exposed to 100 µg/ml concentration of different endophytic fungal extracts namely IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5, IIIM6 and 5-FU (20 µM) as positive control in the culture medium and incubated for 48 h. The cells were fixed with ice-cold TCA for 1 h at 4 °C and plates were washed 5 times in distilled water and dried in air. Further, to each well of the dry 96-well plates 0.4 % sulphorhodamine (SRB) solution was added and allowed to stain at room temperature for 30 min. The plates were washed quickly with 1 % v/v acetic acid to remove the unbound SRB dye. The bound SRB dye was solubilised by adding 10 mM unbuffered tris base (pH 10.5) to each 96 well plate on a shaker platform and plates were read at 540 nm [15].
In-vivo anticancer assay
In-vivo anticancer activity against Ehrlich Ascites Carcinoma (EAC) For propagation of EAC cells, ascetic fluid from an animal bearing 8–10 days old EAC was withdrawn and diluted with normal saline. Cell number per ml of diluted ascetic fluid was determined with the help of Neubauer’s chamber and the volume of ascetic fluid containing 1 × 107 cells was arrived at. This volume of ascetic fluid was injected intraperitoneally in non-inbred Swiss mice (4–5 nos.). When the ascites grew 8–10 days old, again peritoneal fluid was collected and EAC cells were transplanted in the peritoneal cavity of fresh non-inbred Swiss mice. The experimental animals (Non-inbred Swiss mice) weighing 18–23 g were used for experimentation. All the animals used in a single experiment were of the same sex and clinically free Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
DrugRes/2014-12-0933/21.5.2015/MPS
from any disease. The number of animals and the experimental protocols used in the present study were approved by the Institutional Animal Ethics Committee of IIIM, Jammu. For induction of tumor in experimental animals, the ascetic fluid collected from an animal harbouring 8–10 days old EAC was diluted with normal saline in such a way that 0.2 ml of fluid contained 1 × 107 EAC cells. All animals selected for conducting the experiment were injected intraperitoneally with 0.2 ml of ascetic fluid containing 1 × 107 EAC cells on day 0. On day 1, all animals injected with EAC cells were randomized and divided in different treatment and control groups. Tumor bearing control group contained 15 animals and all other groups (treatment and positive control) contained 7 animals each. Animals in each group were weighed and an average body weight of animals in each group was worked out. Based on the body weight, test drugs were prepared for 4 days’ daily administration in such a way that each dose was contained in 0.2 ml volume. Positive control group was treated with 5-Fluorouracil (20 mg/ kg i/p). Tumor bearing control group was administered normal saline (0.2 ml i/p). Animals in the treatment and control groups were treated with respective test drugs at a fixed time (2.00 PM) from day 1–4. On day 5, animals in each group were again weighed and based on the average body weight for each group, test drugs were prepared for the next 5 days. Animals in the treated and control groups were treated with respective test solution at a fixed time during next day 5–9. The experiment was evaluated on day 12. Animals in each group were sacrificed by cervical dislocation. The ascetic fluid from each animal was collected in a pre-weighed graduated centrifuge tube with the help of funnel. Thus, volume and weight of ascetic fluid from each animal was recorded. The number of tumor cells in ascetic fluid was counted with the help of neubauer’s chamber and the total number of tumor cells present in the ascetic fluid of each animal was arrived at. The per cent tumor growth inhibition was calculated as follows. Percent tumor growth inhibition =
Av. no. of tumor cells in control group × 100 Av. no. of cells in treated group
Extraction of Genomic DNA
The endophytic fungal strains were grown in potato dextrose broth (PDB) at 28 °C in an incubator shaker at shaking for 7–10 days. The mycelia from the endophytic fungal strains were collected freeze-dried and the total genomic DNA was extracted by the CTAB (Cetyl Trimethyl Ammonium Bromide) method [16].
Characterization and phylogenetic analysis by partial ITS1-5.8S-ITS2 regions
Identification of the fungal endophytes was based on their internal transcribed spacer ribosomal DNA (ITS rDNA) sequences. A pair of primers ITS4 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS5 (5′-TCC TCC GCT TAT TGA TAT GC-3′) was used for ITS region amplification, using the polymerase chain reaction (PCR). The PCR products were purified, visualized on 1 % agarose gel, sequenced from both directions using ABI Prism 377 Genetic Analyzer (Perkin-Elmer). The resulting sequences were assembled into contigs and basecalls and were edited manually. The consensus sequences were subjected to BLAST searches through NCBI Gen Bank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for subsequent identification and sequence homology with closely related organisms. All the endophytic fungal sequences
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Original Article
DrugRes/2014-12-0933/21.5.2015/MPS
Original Article
Results
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Isolation of the fungal endophytes
From all the 4 selected medicinal plants, total 28 fungal endophytes have been isolated. Based on the morphology of the endophytes 14 different endophytes have been selected for different biological activities. The selected endophytes are IIIM1, IIIM2, IIIM3, IIIM4, IIIM5, IIIM6, IIIM7, IIIM8, IIIM9, IIIM10, IIIM11, IIIM12, IIIM13, and IIIM14. The 7 potent endophytic fungal strains were characterized at molecular level using the ITS15.8 S-ITS2 universal primers.
Molecular identification and characterization of endophytic fungi
The bioactive strains were identified up to genus level using ITS1-5.8 S-ITS2 approach. A total number of 7 strains were selected for the molecular and phylogenetic analysis among the 14 endophytic fungi. The acquisition of ITS1-5.8 S-ITS2 sequence data and their analyses showed diverse taxonomic affinities among the isolated endophytes. Among the identified fungi, Pichia kudriavzevii (query cover 100 %, max identity 99 %), Fusarium oxysporum (query cover 97 %, max identity 99 %), Mucor circinelloide (query cover 99 %, max identity 99 %), Trametes versicolor (query cover 94 %, max identity 95 %), Polyporales sp. (query coverage 100 %, max identity 99 %), Bjerkandera adusta (query coverage 98 %, max identity 99 %), Fusarium tricinctum (query coverage 100 %, max identity 100 %). The sequences was submitted to NCBI Gene Bank and accession numbers assigned to them are; KC741444, KC831587, KC831588, KC831589, KC83 1590, KC831591, and KC831592. The phylogenetic relationship
Fig. 1 Phylogenetic tree generated by the unweighted pair group method with arithmetic means (UPGMA) of simple matching coefficient based on the data amplified from the 7 fungal endophytic isolates using ITS1-5.8 S-ITS2 primers.
tree was constructed and the phylogenetic relationship showed by the identified endophytes is shown in ● ▶ Fig. 1.
In-vitro anticancer activity of the endophytic extracts
Cytotoxicity assay based on SRB was performed against panel of cancer cell lines using series of extracts i. e., IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6. In order to determine the effect of different endophytic extracts (IIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6) on cell proliferation, different human cancer cell lines i. e., colon cancer (caco-2), Leukemia (THP-1), Lung (A549), Neuroblastoma (IMR-32), Breast (MCF-7) and PC-3 (Prostrate) were treated with these endophytic extracts at indicated concentrations (100 µg/ml) for 48 h. In the present study, IIIM8, IIIM2, IIIM3, IIIM7 produced potent inhibition of cell proliferation on caco-2, THP-1, Lung A549, MCF-7 and PC-3 cancer ▶ Table 1). Moreover, IIIM4, IIIM5 and IIIM6 showed cell line ( ● potent inhibition on A549 (lung) only. These results depicted that IIIM8, IIIM2, IIIM3, and IIIM7 showed potent inhibition against all cancer cell lines except IMR-32 as reflected by relative percentage growth inhibition.
In-vivo anticancer activity of the endophytic extracts
The in-vivo anticancer activity of the different endophytic extracts was assessed according to the standard operating procedures. Among the tested extracts against the Ehrlich Ascites Carcinoma mouse modal IIIM2 showed the 58.95 % tumor growth inhibition followed by IIIM8 46.16 %, IIIM3 36.22 % and IIIM7 26.39 % as shown in ● ▶ Table 2.
Discussion
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In the search for new bioactive compounds for potential applications, scientists have turned their attention to natural products from plants and microorganisms. High success rates have been achieved with fungi [17]. A comprehensive study has indicated that 51 % of bioactive substances isolated from endophytic fungi were previously unknown [18]. Hence the endophytic fungi are expected to be a potential source for new natural bioactive molecules. During the present study we evaluated the cytotoxicity effect of IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6 against panel of human cancer cell lines in-vitro which includes colon (caco-2), leukemia (THP-1), lung (A549), neuroblastoma (IMR-32), breast (MCF-7) and prostrate (PC-3). Notably, we found that 4 extracts namely, IIIM8, IIIM2, IIIM3 and IIIM7 showed concentration dependent inhibition of cell growth against all cancer cell lines tested. The maximum growth inhibition was observed with
Table 1 In vitro Anticancer activity of the endophytic extracts. Tissue type
Colon
Prostate
Lung
Leukemia
Neuroblastoma
Breast
Cell line type
Caco-2
PC-3
A549
THP-1
IMR-32
MCF-7
67.00 ± 1.00 22.00 ± 2.00 24.00 ± 2.00 52.00 ± 1.00 50.00 ± 1.00 37.00 ± 1.00 10.00 ± 1.00 89.00 ± 1.00
53.00 ± 2.00 50.00 ± 3.00 20.00 ± 2.00 22.00 ± 1.00 31.00 ± 1.00 48.00 ± 2.00 73.00 ± 2.00 82.00 ± 2.00
Sr. No.
Code
Conc (µg/ml)
1 2 3 4 5 6 7 8
IIIM2 IIIM3 IIIM4 IIIM5 IIIM6 IIIM7 IIIM8 5-FU
100.00 100.00 100.00 100.00 100.00 100.00 100.00 20 µM
% GROWTH INHIBITION 100.00 ± 0.00 76.00 ± 1.00 32.00 ± 2.00 13.00 ± 1.00 10.00 ± 1.00 72.00 ± 2.00 100.00 ± 0.00 20.00 ± 1.00
80.00 ± 1.00 67.00 ± 1.00 21.00 ± 1.00 17.00 ± 1.00 34.00 ± 1.00 55.00 ± 2.00 88.00 ± 1.00 76.00 ± 1.00
75.00 ± 2.00 58.00 ± 2.00 60.00 ± 3.00 56.00 ± 2.00 56.00 ± 1.00 70.00 ± 2.00 71.00 ± 3.00 –
79.00 ± 1.00 60.00 ± 1.00 15.00 ± 1.00 17.00 ± 1.00 48.00 ± 2.00 56.00 ± 2.00 88.00 ± 2.00 73.00 ± 2.00
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
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were deposited in NCBI Gen Bank and the accession number was assigned to all of them. The phylogenetic relationship was ascertained in between the identified sequences.
DrugRes/2014-12-0933/21.5.2015/MPS
Original Article
Table 2 In vivo anticancer activity of the endophytic extracts. Treatment
IIIM2 IIIM3 IIIM7 IIIM8 5-FU Control
Dose (mg/Kg i. p.)
60 100 100 100 20 0.2 ml N. S.
Day 12 AV * ascitic
AW # ascitic
AN € tumor
% Tumor cell
% Tumor growth
fluid (ml)
fluid (g)
cells ( × 107)
growth
inhibition
8.92 ± 1.13 3.41 ± 0.30 6.37 ± 1.05 6.9 ± 1.34 1 ± 0.64 9.36 ± 0.84
8.48 ± 1.11 2.96 ± 0.35 6.37 ± 1.09 6.87 ± 1.35 0.9 ± 0.90 9.25 ± 0.87
111.95 ± 21.66 173.91 ± 9.85 200.72 ± 21.82 146.82 ± 23.63 12.76 ± 8.28 272.69 ± 30.40
41.05 63.78 73.61 53.84 4.67 100
58.95 36.22 26.39 46.16 95.32 0
Mortality 0/7 0/7 0/7 0/7 0/7 0/15
IIIM8 followed by IIIM2, IIIM3, and IIIM7. However, neither of these extracts showed any activity against the cancer cell line IMR-32 when tested at a concentration of 100 µg/ml. Further the 4 active extracts were tested against Ehrlich Ascites Carcinoma mouse model for in-vivo anticancer activity. 2 among all 4 extracts showed considerable in-vivo anticancer activity against the Ehrlich Ascites Carcinoma. The bioactive endophytic strains were identified by the molecular biology approach and sequences were submitted to NCBI Gene Bank. The accession numbers were assigned to all the identified sequences and the phylogenetic relationship was ascertained among the selected sequences. To the best of our knowledge, for the first time we reported the both in-vitro and in-vivo anticancer activities of endophytic fungal isolates of high value medicinal plants of Kashmir Valley, India. The reported fungal endophytes could be used as an appropriate source to produce anticancer agents. Based on the study carried out it can be concluded that the 2 elite endophytic fungal strains, Polyporales spp., and Bjerkandera adusta have the potential to act as a source of new compounds active against a panel of human cancer cell lines. IIIM2 endophytic strain was isolated from Rheum emodi, a Chinese medicinal herb, source of emodin (EMD) that has been widely used for treatment of various ailments and the anticancer activity of EMD [19,20,21]. Emodin could reverse the multi-drug resistance in MCF-7/Adr cells and down regulate ERCC1 protein expression [22]. In addition several other biological activities such as laxative, diuretic, antioxidant, anti-cancer and in vivo inhibitory effects towards P388 leukemia in mice are also reported [23, 24, 25, 26]. IIIM8 isolated from Dioscoria deltoidea is a source of diosgenin, a steroid could significantly inhibit the growth of sarcoma 180 tumour cells in-vivo [27]. The anti-tumor mode of action of diosgenin has been demonstrated via modulation of multiple cell signaling events involving critical molecular candidates associated with growth, differentiation, apoptosis and oncogenesis [28]. This strongly supports our results that the endophytes from these could be appropriate source of anticancer secondary metabolites and it is also evident from the study that fungal endophytes are a rich source of bioactive compounds [29, 30, 31, 32]. These reports and our results strongly support the view that the endophytic fungi of traditional medicinal plants are promising sources of natural anticancer compounds [33, 34, 35].
Conclusion
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This study provided some data on the fungal endophytic diversity of selected high value medicinal plants from the Kashmir
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
valley and also identified some fungal endophytes with potential pharmaceutical applications. The results of the study suggests that there is huge untapped potential for discovering beneficial, novel endophytic fungal candidates from the untapped flora that is found within the Kashmir valley and therefore further endophyte bioprospecting work needs to be conducted within the valley. The findings also suggests that 2 endophytic fungal strains are potential sources for new natural bioactive molecules with pharmaceutical applications and could serve as alternate sources of anticancer agents.
Acknowledgements
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The authors are thankful to CSIR, New Delhi – India for providing the financial assistance during the entire tenure of this work. We are also thankful to Dr. S. N. Sharma, Plant Systematic and Taxonomy, IIIM – Jammu for identification and authentication of the collected medicinal plants. The first author is also thankful to Dr. Edson Panganayi Sibanda, Principal Research Scientist Scientific and Industrial Research and Development Centre (SIRDC), Zimbabwe and Prof. Avinash B. Ade, Department of Botany, University of Pune, Maharashtra, India for revision of the manuscript. The IIIM IP NO. of this manuscript is (IIIM/1746/2014).
Conflict of Intrest
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The authors declare that there is no conflict of intrest among authors.
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DrugRes/2014-12-0933/21.5.2015/MPS
Original Article
Cytotoxic Potential and Molecular Characterization of Fungal Endophytes from Selected High Value Medicinal Plants of the Kashmir Valley – India
Authors
R. A. Dar1, P. H. Qazi1, I. Saba1, S. A. Rather1, Z. A. Wani2, A. K. Qazi2, A. A. Shiekh1, A. Manzoor1, A. Hamid2, D. M. Modae2
Affiliations
1
Key words ▶ ehrlich ascites carcinoma ● ▶ SRB ● ▶ secondary metabolites ●
Abstract
Biotechnology Division, CSIR – Indian Institute of Integrative Medicine, Sanatnagar – Srinagar, Kashmir, India Cancer Pharmacology Division, CSIR – Indian Institute of Integrative Medicine, Jammu – Tawi, India
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The present study explores the fungal endophytes from selected high value medicinal plants to check their activities at in-vitro and in-vivo level. The in-vitro cytotoxicity of selected endophytes revealed potent growth inhibition against human cancer cell lines of leukemia (THP-1), lung (A549), prostate (PC-3), colon (Caco-2), neuroblastoma (IMR-32) and breast (MCF-7) at a concentration of 100 µg/ml. Among them the endophytic strains i. e., IIIM2, IIIM3, IIIM7 and IIIM8 showed most significant growth inhibition
Introduction
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received 28.12.2014 accepted 05.05.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1550042 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence P. H. Qazi Biotechnology Division Indian Institute of Integrative Medicine (CSIR) Sanatnagar – Srinagar 190005 Kashmir India Tel.: + 911/94/2431 255 Fax: + 911/94/2430 779 [email protected]
In the past few decades, scientists have realized that plants act as a reservoir of unexplored potential beneficial microorganisms known as endophytes. These endophytic microbes can produce secondary metabolites such as enzymes, growth hormones, antimicrobial, antifungal or anticancer substances [1, 2]. The discovery of taxol producing endophytic fungus (Taxomyces andreanae) derived from Pacific yew (Taxus brevifolia) was unexpected [3]. Fungal endophytes living in association with medicinal plants have received increasing attention after the discovery that the endophytic fungus Taxomyces andreanae and Tubercularia spp., recovered from Taxus brevifolia and Taxus mairei respectively, also produce the anticancer drug paclitaxel (Taxol) [4, 5]. The discovery that rare, valuable plant products might also be produced by their endophytic microorganisms is of special pharmacological interest [6]. Various workers around the globe have demonstrated that the endophytic fungal communities living within medicinal plant tissues produce a wide range of metabolites with different biological activities [7, 8, 9] that may be
against colon (Caco-2), prostate (PC-3), lung (A549) and leukemia (THP-1) cancer cell lines. At the in-vivo level maximum (58.95 %) tumor growth inhibition was documented with the extract of IIIM2 against Ehrlich Ascites Carcinoma mouse modal. All the potent fungal endophytic strains were characterized using ITS 4 and ITS 5 region sequencing and phylogenetic analysis was ascertained among them. This paper confirms the 2 elite endophytic fungal strains, IIIM2 and IIIM8, have the potential to act as a source of new anticancer compounds.
useful as scaffolds for the development of new drugs. As per reports a wide variety of natural products can be obtained from endophytic microbes [10, 11]. Endophytes associated with the plants producing bioactive natural products also produce the same natural products due to host-endophyte co-evolution [12]. Endophytes have been intensively studied in several unexplored environments around the world. There is a huge interest across the world to determine the biodiversity of endophytic mycoflora within the plant kingdom with the hope of discovering unique endophytes that produce novel compounds that are beneficial to man [13]. To harness the potential of hidden treasure, endophytes have been isolated from indigenous plant species collected from the Kashmir valley in the Indian state of Jammu & Kashmir. In the present study an attempt was made to isolate endophytic fungi from selected high value medicinal plants of Kashmir valley and assessed their in-vitro and in-vivo antitumor potentials. The aim of the study was to assess potential of the endophytic fungi as sources of anticancer compounds which can be used as drugs in the treatment of cancer.
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
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2
Materials and Methods
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Collection and identification of plant material
The selected high value medicinal plants were collected from 2 regions, Sonamarg, Jammu and Kashmir, India (Latitude: 34 °18’17.00 N, Longitude: 75 °17’08.00 E, Altitude: 8711 feet) and the field station of CSIR-Indian Institute of Integrative Medicine, Sanantnagar, Kashmir – India (Latitude: 34 °01’54.29 N, Longitude: 74 °47’53.12 E, Altitude: 5220 feet). 4 types of plants in 5 replicates were collected randomly from each region by individual and group visits (Rheum emodi wall. ex Meissn and Hypercum perforatum L. from Sonamarg and Diocoria deltodia Wall. and Artemisia annua L. from the field station of IIIM Sanantnagar). A voucher specimen of each plant was submitted to the Division of Plant Systematic and Taxonomy, CSIR-IIIM – Jammu for identification and authentication.
Isolation, purification and extraction of different endophytic strains
The endophytic fungal strains were isolated, purified and preserved in glycerol [14]. For extraction, the endophytic fungal strains were grown on potato dextrose broth at 180 rpm, 28 °C for 10–15 days in an incubator shaker (New Brunswick, USA). The grown culture of each endophyte was homogenized with 10 % methanol under ultra sonicator. The fermented broth was then extracted with dichloromethane 3 times after which Na2SO4 (40 μg/ml, Merck) was added to further remove the aqueous layer within the mixture. Further the mixture was transferred to a round bottom flask and dried using a rotary evaporator and weighed to constitute the crude broth extract.
In-vitro cytotoxicity assay
The in-vitro cell cytotoxicity was checked by sulphorhodamine B (SRB) assay. In this assay cell suspension of optimum cell density was seeded and exposed to 100 µg/ml concentration of different endophytic fungal extracts namely IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5, IIIM6 and 5-FU (20 µM) as positive control in the culture medium and incubated for 48 h. The cells were fixed with ice-cold TCA for 1 h at 4 °C and plates were washed 5 times in distilled water and dried in air. Further, to each well of the dry 96-well plates 0.4 % sulphorhodamine (SRB) solution was added and allowed to stain at room temperature for 30 min. The plates were washed quickly with 1 % v/v acetic acid to remove the unbound SRB dye. The bound SRB dye was solubilised by adding 10 mM unbuffered tris base (pH 10.5) to each 96 well plate on a shaker platform and plates were read at 540 nm [15].
In-vivo anticancer assay
In-vivo anticancer activity against Ehrlich Ascites Carcinoma (EAC) For propagation of EAC cells, ascetic fluid from an animal bearing 8–10 days old EAC was withdrawn and diluted with normal saline. Cell number per ml of diluted ascetic fluid was determined with the help of Neubauer’s chamber and the volume of ascetic fluid containing 1 × 107 cells was arrived at. This volume of ascetic fluid was injected intraperitoneally in non-inbred Swiss mice (4–5 nos.). When the ascites grew 8–10 days old, again peritoneal fluid was collected and EAC cells were transplanted in the peritoneal cavity of fresh non-inbred Swiss mice. The experimental animals (Non-inbred Swiss mice) weighing 18–23 g were used for experimentation. All the animals used in a single experiment were of the same sex and clinically free Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
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from any disease. The number of animals and the experimental protocols used in the present study were approved by the Institutional Animal Ethics Committee of IIIM, Jammu. For induction of tumor in experimental animals, the ascetic fluid collected from an animal harbouring 8–10 days old EAC was diluted with normal saline in such a way that 0.2 ml of fluid contained 1 × 107 EAC cells. All animals selected for conducting the experiment were injected intraperitoneally with 0.2 ml of ascetic fluid containing 1 × 107 EAC cells on day 0. On day 1, all animals injected with EAC cells were randomized and divided in different treatment and control groups. Tumor bearing control group contained 15 animals and all other groups (treatment and positive control) contained 7 animals each. Animals in each group were weighed and an average body weight of animals in each group was worked out. Based on the body weight, test drugs were prepared for 4 days’ daily administration in such a way that each dose was contained in 0.2 ml volume. Positive control group was treated with 5-Fluorouracil (20 mg/ kg i/p). Tumor bearing control group was administered normal saline (0.2 ml i/p). Animals in the treatment and control groups were treated with respective test drugs at a fixed time (2.00 PM) from day 1–4. On day 5, animals in each group were again weighed and based on the average body weight for each group, test drugs were prepared for the next 5 days. Animals in the treated and control groups were treated with respective test solution at a fixed time during next day 5–9. The experiment was evaluated on day 12. Animals in each group were sacrificed by cervical dislocation. The ascetic fluid from each animal was collected in a pre-weighed graduated centrifuge tube with the help of funnel. Thus, volume and weight of ascetic fluid from each animal was recorded. The number of tumor cells in ascetic fluid was counted with the help of neubauer’s chamber and the total number of tumor cells present in the ascetic fluid of each animal was arrived at. The per cent tumor growth inhibition was calculated as follows. Percent tumor growth inhibition =
Av. no. of tumor cells in control group × 100 Av. no. of cells in treated group
Extraction of Genomic DNA
The endophytic fungal strains were grown in potato dextrose broth (PDB) at 28 °C in an incubator shaker at shaking for 7–10 days. The mycelia from the endophytic fungal strains were collected freeze-dried and the total genomic DNA was extracted by the CTAB (Cetyl Trimethyl Ammonium Bromide) method [16].
Characterization and phylogenetic analysis by partial ITS1-5.8S-ITS2 regions
Identification of the fungal endophytes was based on their internal transcribed spacer ribosomal DNA (ITS rDNA) sequences. A pair of primers ITS4 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS5 (5′-TCC TCC GCT TAT TGA TAT GC-3′) was used for ITS region amplification, using the polymerase chain reaction (PCR). The PCR products were purified, visualized on 1 % agarose gel, sequenced from both directions using ABI Prism 377 Genetic Analyzer (Perkin-Elmer). The resulting sequences were assembled into contigs and basecalls and were edited manually. The consensus sequences were subjected to BLAST searches through NCBI Gen Bank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for subsequent identification and sequence homology with closely related organisms. All the endophytic fungal sequences
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Original Article
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Original Article
Results
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Isolation of the fungal endophytes
From all the 4 selected medicinal plants, total 28 fungal endophytes have been isolated. Based on the morphology of the endophytes 14 different endophytes have been selected for different biological activities. The selected endophytes are IIIM1, IIIM2, IIIM3, IIIM4, IIIM5, IIIM6, IIIM7, IIIM8, IIIM9, IIIM10, IIIM11, IIIM12, IIIM13, and IIIM14. The 7 potent endophytic fungal strains were characterized at molecular level using the ITS15.8 S-ITS2 universal primers.
Molecular identification and characterization of endophytic fungi
The bioactive strains were identified up to genus level using ITS1-5.8 S-ITS2 approach. A total number of 7 strains were selected for the molecular and phylogenetic analysis among the 14 endophytic fungi. The acquisition of ITS1-5.8 S-ITS2 sequence data and their analyses showed diverse taxonomic affinities among the isolated endophytes. Among the identified fungi, Pichia kudriavzevii (query cover 100 %, max identity 99 %), Fusarium oxysporum (query cover 97 %, max identity 99 %), Mucor circinelloide (query cover 99 %, max identity 99 %), Trametes versicolor (query cover 94 %, max identity 95 %), Polyporales sp. (query coverage 100 %, max identity 99 %), Bjerkandera adusta (query coverage 98 %, max identity 99 %), Fusarium tricinctum (query coverage 100 %, max identity 100 %). The sequences was submitted to NCBI Gene Bank and accession numbers assigned to them are; KC741444, KC831587, KC831588, KC831589, KC83 1590, KC831591, and KC831592. The phylogenetic relationship
Fig. 1 Phylogenetic tree generated by the unweighted pair group method with arithmetic means (UPGMA) of simple matching coefficient based on the data amplified from the 7 fungal endophytic isolates using ITS1-5.8 S-ITS2 primers.
tree was constructed and the phylogenetic relationship showed by the identified endophytes is shown in ● ▶ Fig. 1.
In-vitro anticancer activity of the endophytic extracts
Cytotoxicity assay based on SRB was performed against panel of cancer cell lines using series of extracts i. e., IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6. In order to determine the effect of different endophytic extracts (IIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6) on cell proliferation, different human cancer cell lines i. e., colon cancer (caco-2), Leukemia (THP-1), Lung (A549), Neuroblastoma (IMR-32), Breast (MCF-7) and PC-3 (Prostrate) were treated with these endophytic extracts at indicated concentrations (100 µg/ml) for 48 h. In the present study, IIIM8, IIIM2, IIIM3, IIIM7 produced potent inhibition of cell proliferation on caco-2, THP-1, Lung A549, MCF-7 and PC-3 cancer ▶ Table 1). Moreover, IIIM4, IIIM5 and IIIM6 showed cell line ( ● potent inhibition on A549 (lung) only. These results depicted that IIIM8, IIIM2, IIIM3, and IIIM7 showed potent inhibition against all cancer cell lines except IMR-32 as reflected by relative percentage growth inhibition.
In-vivo anticancer activity of the endophytic extracts
The in-vivo anticancer activity of the different endophytic extracts was assessed according to the standard operating procedures. Among the tested extracts against the Ehrlich Ascites Carcinoma mouse modal IIIM2 showed the 58.95 % tumor growth inhibition followed by IIIM8 46.16 %, IIIM3 36.22 % and IIIM7 26.39 % as shown in ● ▶ Table 2.
Discussion
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In the search for new bioactive compounds for potential applications, scientists have turned their attention to natural products from plants and microorganisms. High success rates have been achieved with fungi [17]. A comprehensive study has indicated that 51 % of bioactive substances isolated from endophytic fungi were previously unknown [18]. Hence the endophytic fungi are expected to be a potential source for new natural bioactive molecules. During the present study we evaluated the cytotoxicity effect of IIIM8, IIIM2, IIIM3, IIIM7, IIIM4, IIIM5 and IIIM6 against panel of human cancer cell lines in-vitro which includes colon (caco-2), leukemia (THP-1), lung (A549), neuroblastoma (IMR-32), breast (MCF-7) and prostrate (PC-3). Notably, we found that 4 extracts namely, IIIM8, IIIM2, IIIM3 and IIIM7 showed concentration dependent inhibition of cell growth against all cancer cell lines tested. The maximum growth inhibition was observed with
Table 1 In vitro Anticancer activity of the endophytic extracts. Tissue type
Colon
Prostate
Lung
Leukemia
Neuroblastoma
Breast
Cell line type
Caco-2
PC-3
A549
THP-1
IMR-32
MCF-7
67.00 ± 1.00 22.00 ± 2.00 24.00 ± 2.00 52.00 ± 1.00 50.00 ± 1.00 37.00 ± 1.00 10.00 ± 1.00 89.00 ± 1.00
53.00 ± 2.00 50.00 ± 3.00 20.00 ± 2.00 22.00 ± 1.00 31.00 ± 1.00 48.00 ± 2.00 73.00 ± 2.00 82.00 ± 2.00
Sr. No.
Code
Conc (µg/ml)
1 2 3 4 5 6 7 8
IIIM2 IIIM3 IIIM4 IIIM5 IIIM6 IIIM7 IIIM8 5-FU
100.00 100.00 100.00 100.00 100.00 100.00 100.00 20 µM
% GROWTH INHIBITION 100.00 ± 0.00 76.00 ± 1.00 32.00 ± 2.00 13.00 ± 1.00 10.00 ± 1.00 72.00 ± 2.00 100.00 ± 0.00 20.00 ± 1.00
80.00 ± 1.00 67.00 ± 1.00 21.00 ± 1.00 17.00 ± 1.00 34.00 ± 1.00 55.00 ± 2.00 88.00 ± 1.00 76.00 ± 1.00
75.00 ± 2.00 58.00 ± 2.00 60.00 ± 3.00 56.00 ± 2.00 56.00 ± 1.00 70.00 ± 2.00 71.00 ± 3.00 –
79.00 ± 1.00 60.00 ± 1.00 15.00 ± 1.00 17.00 ± 1.00 48.00 ± 2.00 56.00 ± 2.00 88.00 ± 2.00 73.00 ± 2.00
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
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were deposited in NCBI Gen Bank and the accession number was assigned to all of them. The phylogenetic relationship was ascertained in between the identified sequences.
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Original Article
Table 2 In vivo anticancer activity of the endophytic extracts. Treatment
IIIM2 IIIM3 IIIM7 IIIM8 5-FU Control
Dose (mg/Kg i. p.)
60 100 100 100 20 0.2 ml N. S.
Day 12 AV * ascitic
AW # ascitic
AN € tumor
% Tumor cell
% Tumor growth
fluid (ml)
fluid (g)
cells ( × 107)
growth
inhibition
8.92 ± 1.13 3.41 ± 0.30 6.37 ± 1.05 6.9 ± 1.34 1 ± 0.64 9.36 ± 0.84
8.48 ± 1.11 2.96 ± 0.35 6.37 ± 1.09 6.87 ± 1.35 0.9 ± 0.90 9.25 ± 0.87
111.95 ± 21.66 173.91 ± 9.85 200.72 ± 21.82 146.82 ± 23.63 12.76 ± 8.28 272.69 ± 30.40
41.05 63.78 73.61 53.84 4.67 100
58.95 36.22 26.39 46.16 95.32 0
Mortality 0/7 0/7 0/7 0/7 0/7 0/15
IIIM8 followed by IIIM2, IIIM3, and IIIM7. However, neither of these extracts showed any activity against the cancer cell line IMR-32 when tested at a concentration of 100 µg/ml. Further the 4 active extracts were tested against Ehrlich Ascites Carcinoma mouse model for in-vivo anticancer activity. 2 among all 4 extracts showed considerable in-vivo anticancer activity against the Ehrlich Ascites Carcinoma. The bioactive endophytic strains were identified by the molecular biology approach and sequences were submitted to NCBI Gene Bank. The accession numbers were assigned to all the identified sequences and the phylogenetic relationship was ascertained among the selected sequences. To the best of our knowledge, for the first time we reported the both in-vitro and in-vivo anticancer activities of endophytic fungal isolates of high value medicinal plants of Kashmir Valley, India. The reported fungal endophytes could be used as an appropriate source to produce anticancer agents. Based on the study carried out it can be concluded that the 2 elite endophytic fungal strains, Polyporales spp., and Bjerkandera adusta have the potential to act as a source of new compounds active against a panel of human cancer cell lines. IIIM2 endophytic strain was isolated from Rheum emodi, a Chinese medicinal herb, source of emodin (EMD) that has been widely used for treatment of various ailments and the anticancer activity of EMD [19,20,21]. Emodin could reverse the multi-drug resistance in MCF-7/Adr cells and down regulate ERCC1 protein expression [22]. In addition several other biological activities such as laxative, diuretic, antioxidant, anti-cancer and in vivo inhibitory effects towards P388 leukemia in mice are also reported [23, 24, 25, 26]. IIIM8 isolated from Dioscoria deltoidea is a source of diosgenin, a steroid could significantly inhibit the growth of sarcoma 180 tumour cells in-vivo [27]. The anti-tumor mode of action of diosgenin has been demonstrated via modulation of multiple cell signaling events involving critical molecular candidates associated with growth, differentiation, apoptosis and oncogenesis [28]. This strongly supports our results that the endophytes from these could be appropriate source of anticancer secondary metabolites and it is also evident from the study that fungal endophytes are a rich source of bioactive compounds [29, 30, 31, 32]. These reports and our results strongly support the view that the endophytic fungi of traditional medicinal plants are promising sources of natural anticancer compounds [33, 34, 35].
Conclusion
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This study provided some data on the fungal endophytic diversity of selected high value medicinal plants from the Kashmir
Dar RA et al. Cytotoxic Activity of Fungal Endophytes … Drug Res
valley and also identified some fungal endophytes with potential pharmaceutical applications. The results of the study suggests that there is huge untapped potential for discovering beneficial, novel endophytic fungal candidates from the untapped flora that is found within the Kashmir valley and therefore further endophyte bioprospecting work needs to be conducted within the valley. The findings also suggests that 2 endophytic fungal strains are potential sources for new natural bioactive molecules with pharmaceutical applications and could serve as alternate sources of anticancer agents.
Acknowledgements
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The authors are thankful to CSIR, New Delhi – India for providing the financial assistance during the entire tenure of this work. We are also thankful to Dr. S. N. Sharma, Plant Systematic and Taxonomy, IIIM – Jammu for identification and authentication of the collected medicinal plants. The first author is also thankful to Dr. Edson Panganayi Sibanda, Principal Research Scientist Scientific and Industrial Research and Development Centre (SIRDC), Zimbabwe and Prof. Avinash B. Ade, Department of Botany, University of Pune, Maharashtra, India for revision of the manuscript. The IIIM IP NO. of this manuscript is (IIIM/1746/2014).
Conflict of Intrest
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The authors declare that there is no conflict of intrest among authors.
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