Background
Taxol, also known as paclitaxel, is a complex diterpenoid compound originally reported from bark of the Pacific yew tree,
Taxus brevifolia [
1] and later reported in other yew species [
2]. Taxol is widely used against breast, ovarian and lung cancers [
3,
4]. Acute supply crisis prevails for paclitaxel as chemotherapeutic drugs since its concentration in yew bark is exceedingly low and extraction process is complicated and expensive [
5]. Besides, overexploitation of yew bark has led to serious diminishing of
Taxus forests [
6,
7]. Therefore, several approaches have been utilized for increasing taxol accessibility and finding alternative sources through chemical synthesis, tissue and cell cultures of the
Taxus spp. [
8‐
12]. However, the efforts failed to increase the yield of taxol, improve the complicated process and decrease the cost [
8,
11,
13]. This finally compelled the researchers to explore the microbial world. Microbial fermentation with the benefits of optimization of fermentation conditions and co-cultivation offers suitable inexpensive method of choice to increase yield of taxol production. In the microorganisms, taxol was first reported from an endophytic fungus
Taxomyces andreanae isolated from the inner bark of
Taxus brevifolia [
14]. A large number of taxol-producing endophytic fungi such as
Pestalotiopsis microspora,
Taxomyces andreanae,
Fusarium spp.,
Alternaria sp. and
Tubercularia sp. have been reported from
Taxus plants since then [
15‐
20]. Additionally, several reports have shown that non-
Taxus plants also harbour taxol-producing endophytic fungi such as
Periconia sp.,
Bartalinia robilldoides and
Pestalotiopsis guepinii [
21‐
23]. A total of 100 reports of endophytic fungi belonging to 72 fungal species from 32 different host plants have been reported so far for taxol production [
24].
Cancer is one of the leading causes of death in the world [
25] and hepatocellular carcinoma (HCC) is the fifth most common cancers worldwide and the third most common reason for cancer-related mortality [
26]. Surgical resection and liver transplantation are inefficient for advanced HCC [
27,
28]. Hence, it is imperative to develop new therapeutic drugs with high efficacy and low toxicity for HCC. Apoptosis, a programmed cell suicide, is usually a physiological event that does not induce inflammation [
29]. Therefore, apoptosis induction is considered a desired therapeutic goal in cancer treatment to reduce possible adverse side effects [
30]. Many studies have demonstrated apoptosis by taxol treatment in diverse cancer cells including breast cancer, glioblastoma, hepatoma and ovarian cancer. Taxol triggers apoptosis by diverse pro-apoptosis stimuli converging on mitochondria, causing mitochondrial depolarization and caspase enzymes activation eventually leading to apoptotic cell death [
31‐
38]. In the course of continuous research on plant-fungus associations and in search of novel bioactive secondary metabolites from endophytic cultures, a taxol derivative, EDT obtained from an endophytic fungus
P. microspora associated with
T. mucronatum is being reported herewith. It is the first studies to report EDT from a microbial source. We also report characterization and comparison of anti-proliferative and apoptosis inducing activity of EDT in hepatocellular carcinoma cells (HepG2), as well as investigate the molecular mechanisms triggering apoptosis.
Methods
Isolation and identification of endophytic fungi from T. mucronatum
The fungus used in this study was one of 27 endophytic fungi isolated from the inner bark of
Taxodium mucronatum obtained in Ootacamund, South East India. The voucher specimen was deposited at Madras University Herbaria and Culture Collection in Centre for Advanced Studies in Botany, Chennai with accession number MUBL1013. The
T. mucronatum bark was cut into pieces (~0.5 × 0.5 × 0.5 cm) and treated with 70% (
v/v) ethanol, washed with sterilized water and the outer bark removed with a sterilized sharp blade. Small pieces of inner bark were placed on the surface of PDA medium supplemented with 150 mg L
−1 chloramphenicol in Petri plates and incubated at 26 ± 1 °C in 12 h light/dark chamber. After several days, fungi were observed growing from the inner bark fragments in the plates. Individual hyphal tips of the various fungi were removed from the agar plates, placed on new PDA medium and incubated at 26 ± 1 °C for at least 2 weeks. Fungus culture was checked for purity and transferred to fresh agar plate by the hyphal tip method [
15]. Fungus was identified based on the morphology of the fungal culture, the mechanism of spore production and the characteristics of the spores [
39]. For molecular identification, DNA extraction and ITS PCR was followed as described earlier [
40]. The universal primers ITS1 and ITS4 were used for amplification. The PCR product was sequenced in an AVI377 automated DNA sequencer. The ten most similar sequences in Genbank were found for sequence by means of BLAST search. Sequences were aligned by using CLUSTAL multiple sequence alignment and gaps were excluded. The most informative sequences were used to construct phylogenetic tree using maximum parsimony by MEGA 4 [
41] and the
Amanita muscaria used for as an out group of organism. The fungal spores and mycelia were preserved in 15% (
v/v) glycerol at −70 °C.
Fermentation, extraction and fungal EDT isolation
The
Pestalotiopsis microspora used in this study was grown in 4 l Erlenmeyer flasks containing 1 l modified M1D medium [
42]. Twelve mycelial agar plugs of 0.5 × 0.5 cm, were used as inoculum. The fungus was grown at 26 ± 1 °C in 12 h light/dark chamber. After 18 days of incubation, the entire culture (1 l) was passed through four layers of cheesecloth. The culture fluid was extracted with two equal volumes of dichloromethane and the organic phase was taken to evaporation under reduced pressure at 40 °C. The residue was dissolved in 1 ml methanol, and subject to TLC on a 0.25 mm (10 × 20 cm) silica gel plate developed in solvent system of chloroform/methanol (7:1,
v/v) with authentic paclitaxel (Sigma, Cat. No. T-7402). After chromatography, the silica gel plate was sprayed with 1% vanillin-sulphuric acid (
w/
v) and visualized under UV fluorescence at 254 and 365 nm for confirmation for taxol/taxanes with appropriate relative front (R
f
) by comparing with the reference paclitaxel.
A total of 14.7 g residue was dissolved in 5 ml methanol suspended with ~ 25 g of silica gel for preparation of slurry (silica gel + sample). Then the dry slurry placed on a 2 × 45 cm column of silica gel (60–120 mesh) equilibrated with chloroform. Elution of the column was performed in a step-wise manner starting with 70 ml 100% chloroform followed by mixtures of chloroform/acetone at 95:5, 90:10, 85:15, 80:20 till 0:100 (v/v). A fraction having the corresponding chromatographic mobility as authentic paclitaxel was found from the 75:25 to 70:30 fractions. These fractions were combined and evaporated to dryness yielding 1.5 g of residues, which was further subjected to a second 1.5 × 30 cm column of silica gel (200–300) and eluted with dichloromethane/acetone (25:75, v/v). This eluted fraction exhibited on R
f
which was identical to reference paclitaxel. Then, the fraction subjected for in vacco at 40 °C yielded yellow powder (11.79 mg).
Spectroscopic analyses for identification of fungal EDT
Nuclear magnetic resonance spectroscopy (NMR) was done on fungal EDT preparation in a JEOL JNM-ECP 600 MHz instrument with the sample dissolved in 100% deuterated methanol. X-ray powder diffraction (XRD) was studied for EDT by coating on the XRD grid and the spectra were recorded by using Philips PW1830 X-ray generator operated at voltage of 40 kV and a current of 30 mA using Cu Kσ−1 radiation. Liquid chromatography-Electrospray ionization-tandem mass spectrometry (LC-ESI-MS) was performed on Thermo Finnigan Survey or HPLC with dual wavelength (UV) detector connected to Thermo LCQ Deca XPMAX-MS platform and analysed by Xcalibur software. The EDT was dissolved in methanol and was injected with a spray flow of 2 μl min−1 and a spray voltage of 2.2 kV. Fourier transform infrared spectroscopy (FTIR) was recorded using Perkin Elmer Spectrum one FTIR over the region 4000-400 cm−1.
Cell lines and culture conditions
HepG2 cells (human liver carcinoma cell line) used for the experiments was obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cells were grown as monolayers in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 1.5 g ml−1 sodium bicarbonate, 2 mM 1−1 glutamine and antibiotics (10,000 U ml−1 pencillin and 10 mg ml−1 streptomycin). Cell stocks were maintained in 75 cm2 tissue culture flasks in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cultures were maintained in the medium until the confluent growth was attained. A cell density of at 1 × 106 cells was maintained at the time of treatment. For all in vitro assays, fungal EDT was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at 1.284 mM concentration, sterilized using a sterile 0.22 μm membrane filter and stored at −20 °C. From the stock, 256.8 μM/ml was taken and diluted with cell culture medium in 2-fold serial dilutions (128.4, 64.2, 32.1, 16.05, 8.02, 4.02, 2.0, 1.0 μM/ml) and 0.5% DMSO maintained as a control.
Cell survival assay
The cytotoxicity of purified fungal EDT was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) colorimetric assay. Briefly, cells (1 × 104 cells/well) were seeded in 96-well plates. After complete adhesion, different concentration of fungal EDT (0–128.4 μM) were added and incubated further for 24 h at 37 °C. The treated cells were then incubated in fresh DMEM medium containing MTT (5 mg mL−1) at 37 °C. After 4 h, the supernatants were discarded carefully and DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured with a microplate reader (Bio-Rad, USA). The cell survival was determined by the MTT test as the percentage of the ratio of the absorbencies of treated and untreated (control cells).
Determination of apoptosis
Apoptosis was determined using acridine orange-ethidium bromide (AO-EBr) dual staining method by microscopically visualizing condensed apoptotic nuclei from normal ones [
43]. HepG2 cells (3 × 10
4) were treated with different concentrations of fungal EDT seeded in 6-well plates and incubated for 24 h. Cells were then stained with 1:1 ratio of AO and EBr. After staining, the cells were immediately visualized using fluorescence microscope (Olympus, CKX41, Japan) at a magnification 40× using 450–490 nm filter. The number of cells showing features of apoptosis was counted as a fraction of the total number of cells present in a field.
Determination of nuclear morphology
The nuclear condensation was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. HepG2 cells (5 × 10
4 cells/well) cultured in 12-well plates were incubated with and without fungal EDT for 24 h. The cells were then fixed with 3.7% (
v/v) paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with DAPI (1 mg ml
−1 in PBS) [
44]. After washing twice with PBS, cells were observed under fluorescence microscope (Olympus, BX51, Japan) at 10× magnification using 485 nm excitation and 535 nm emission filter sets. The apoptotic cells were identified by the presence of highly condensed chromatin or fragmented nuclei.
DNA fragmentation analysis
The DNA fragmentation was studied as described earlier [
44]. HepG2 cells were cultured in 60 mm dishes to 70% confluence prior to drug treatment. The cells (5 × 10
6) were treated with different concentrations of fungal EDT for 24 h. After treatment, the cells were harvested by centrifugation at 1000×
g for 5 min and washed with ice cold PBS. The genomic DNA was extracted from the HepG2 cells using QIAamp DNA Mini Kit (Qiagen, USA), analysed using 0.9% (
w/
v) agarose gel and electrophoresed at 2 V/cm for 16 h. The DNA present in the gels was visualized under UV light after staining with ethidium bromide (1 μg ml
−1) and photographed using gel documentation system (Gene Flash, Syngene, Bioimaging, Kubota 2420, Tokyo).
Apoptosis assay by Propidium iodide (PI) staining
HepG2 cells (3.5 × 106) were seeded in 6-well plates and treated with different concentrations of fungal EDT at 37 °C for 24 h in CO2 incubator. Cells (2 × 106) were fixed in 90% ethanol in PBS at 4 °C for analyzing DNA. After 12 h, the cells were centrifuged at 2000 rpm for 5 mins and the cell pellet was suspended in ice cold PBS. The cell suspension was then treated with propidium iodide along with RNAase (50 μg ml−1) for 30 min at 37 °C in CO2 incubator then stored in the dark at 4 °C. The red fluorescence of the individual cells was measured at an excitation wavelength of 540 nm and an emission wavelength at 610 nm in a FACSCalibur flow cytometer (BD Biosciences, CA). A minimum of 10,000 events were analyzed per sample using CellQuest software.
Measurement of intracellular reactive oxygen species (ROS)
The intracellular ROS generation was detected using an oxidant sensitive non-fluorescent probe DCFH-DA that gets oxidized by intracellular ROS to its fluorescent derivative, dichlorofluorescein (DCF) [
45]. HepG2 cells (8 × 10
6 cells/ml) seeded in 96-well plates were treated with different concentrations of fungal EDT for 24 h, followed by 10 μM DCFH-DA addition and further incubated for 30 min. The cells were washed with PBS to remove the excess dye and measurements were done using spectro-fluorophotometer (Shimadzu, RF-5301PC, USA) with excitation and emission filters set at 485 ± 10 and 530 ± 12.5 nm, respectively. Fluorescent microscopic images were taken using blue filter (450–490 nm) (Olympus, CKX41, Japan).
Western blot analysis
Proteins were isolated from control and fungal EDT-treated cells as described previously [
46]. Bax, Bcl-2, p38 MAPK, PARP and β-actin protein expression was investigated using Western blot analysis. Briefly, cells in 6 well plates were harvested and washed with PBS. Cells were lysed in 100 μl lysis buffer (20 mM Tris–Hcl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 30 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) followed by centrifugation at 1000 g for 5 min at 4 °C. The supernatants (cytosolic fractions) were saved and the pellets solubilized in the same volume of mitochondrial lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 100 μM PMSF, 10 μg/ml leupeptin, 2 μg/ml aprotinin), kept on ice and vortexed for 20 min followed by pelleting at 10,000 g for 10 min at 4 °C and subjected to 12.5% poly acrylamide gel electrophoresis lane. A total volume of 40 μg of protein was loaded per lane. The separated proteins were blotted onto a PVDF membrane by semi-dry transfer (Bio-Rad, USA). After blocking with 5% non-fat milk in TBS, the membranes were then incubated with various antibodies: anti-Bax, anti-Bcl-2, anti-p38 MAPK, anti-PARP and anti-β-actin. The dilutions used were p38 (1:1000), Bcl-2 (1:500), Bax (1:1000), PARP (1:500) and β-actin (1:2000). After primary antibody incubation, the membranes were incubated with secondary antibody at a concentration of 1:2000. Then the membranes were washed with Tris-buffered saline and 0.05% Tween-20 thrice for 10 min interval, after extensive washes in TBST, the bands was visualized by treating the membranes with 3, 30-diaminobenzidine tetrahydrochloride (Western blot detection reagent, Sigma, USA). Densitometry was done using ‘Image J’ analysis software.
Statistical analysis
All data are presented as the means ± standard deviation (SD) for at least three independent experiments, and analyzed for statistical significance using one-way analysis of variance using software SPSS 11.5. A p-value <0.05 was statistically significant.
Discussion
The endophytic fungus,
Pestalotiopsis microspora isolated from non-
Taxus host
Taxodium distichum was first reported in 1996 for taxol production [
17]. At present, many endophytic fungi from
Taxus /non-
Taxus species including
Pestalotiopsis spp. have been recognized for production of taxol and its derivatives [
52]. In the present study, we report a taxol derivative, 7-
epi-10-deacetyltaxol (EDT) isolated from the culture filtrate of
P. microspora as confirmed by TLC and various spectroscopic/spectrometric analyses. To the best of our knowledge, this is the first report of isolation of EDT from only microbial source especially from
P. microspore. P. microspora was identified based on morphological features and molecular data. The morphology of this strain is different from that of other taxol-producing endophytic
Pestalotiopsis spp. [
53‐
55]. The spores of
P. microspora median cells have thicker walls and non-distoseptate [
15‐
17,
47]. In ITS rDNA molecular characterization confirmed the strain as
P. microspora.
The production of EDT by
P. microspora to produce EDT was confirmed by isolation of a compound having chromatographic properties similar to authentic paclitaxel in solvent system chloroform/methanol (7:1,
v/v) which showed a single dark bluish violet spot on TLC when sprayed with vanillin-sulfuric acid reagent and giving the corresponding R
ƒ (0.80) with paclitaxel (0.78) under UV fluorescence at 365 nm. The maximum UV absorption wavelength for fungal EDT was found (λ
max 228) identical to authentic taxol UV absorption at λ
max 229. Our result is coinciding with previously reported plant-derived 7-
epi-10-deacetyltaxol [
56,
57] in
Taxus plants. In ESI-MS, molecular ions at m/z 811 attributing to the (M + H)
+ and confirmed its molecular weight to be 810 for the fungal EDT [
56,
57].
1H and
13C NMR spectra were identical with the authentic taxol spectra and that of spectra previously reported for 7-
epi-10-deacetyltaxol [
49,
50,
58]. Further the functional groups in the fungal EDT analysed using FTIR showed peaks similar to those reported earlier for plant-derived 7-
epi-10-deacetyltaxol [
49]. In addition, to confirm the fungal EDT structure, XRD analysis was performed and the results showed closest match to 7-
epi-10-deacetyltaxol verified by JCPDS computational database. Therefore, these results evidently display the fungal 7-
epi-10-deacetyltaxol (EDT).
To study the anticancer activity of fungal EDT towards liver cancer cells, we used HepG2 cell line. Several studies have previously shown that taxol isolated from plants and endophytic fungi were effective in inhibiting cancer cell proliferation towards ovarian, breast and lung cancers [
3,
4]. In the present study, fungal EDT significantly inhibited the growth of HepG2 cells, with an IC
50 value as 32.1 μM for 24 h treatment, indicating that fungal EDT exhibits strong cytotoxicity against HepG2 cells. Induction of apoptosis is regarded as a novel therapeutic strategy for cancer treatment [
29]. Many anticancer agents have been reported to induce death of tumor cells by triggering apoptosis [
59]. In our results, fungal EDT strongly induced apoptotic cell death in HepG2 cells in a dose-dependent manner as evidenced by EtBr/AO staining, suggesting that the anticancer effect of fungal EDT on HepG2 cells was mediated through the induction of apoptosis. The nuclear shrinkage, chromatin condensation and fragmentation are the main hallmarks of apoptosis; these were observed upon treatment of the cell lines with fungal EDT at 62.4 and 128.4 μM. Inter-nucleosomal DNA fragmentation represents the terminal step in the events leading to apoptosis. Gel electrophoresis of the genomic DNA isolated from fungal EDT-treated HepG2 cells displayed the DNA fragmented. Similar DNA fragmentation was reported on paclitaxel exhibited cell viability on MCF-7 cells [
20]. PI-FACS analysis of fungal EDT-treated HepG2 cells indicated that cell cycle arrested in the G2/M phase with significant effect at 64.2 μM and 128.4 μM arresting cells at 50.08 and 80.16%, respectively.
Several chemotherapeutic agents exert their anticancer effects through inducing the generation of ROS, and the intrinsic apoptotic pathway is especially susceptible to ROS [
60]. In the present study, the production of intracellular ROS increased remarkably in HepG2 cells treated with fungal EDT, while inhibition of ROS production by the control cells significantly decreased the apoptosis, suggesting that fungal EDT-induced apoptosis in HepG2 cells was closely associated with the production of ROS, which may act as upstream signalling molecules to initiate mitochondria-mediated cell apoptosis. ROS is involved in the opening of the mitochondrial permeability transition pore, depolarization of the mitochondrial membrane, and then the release of mitochondrial pro-apoptotic factors in the process of mitochondria mediated apoptosis [
61,
62]. Fungal taxol isolated from
F. solani, exhibited cytotoxicity on JR4-Jurkat cells was related to accumulation of intracellular ROS, reduction of mitochondrial membrane potential, and cell apoptosis [
20]. The convincing genetic/biochemical evidence has accumulated so far to show that taxol-mediated apoptosis solely relies on the intrinsic or the mitochondrial pathway [
37].
Apoptosis is executed through mitochondrial-mediated intrinsic and cell death receptor-mediated extrinsic pathways, both of which converge on the cascade leading to activation of caspase proteases [
63]. In the intrinsic pathway, cytochrome c releases from damaged mitochondria and causes apoptosome-dependent activation of caspase 9. This leads to the activation of the executioner caspase 3 [
64], and eventually proteolytic inactivation of PARP [
62]. In the extrinsic pathway, activated initiator caspase-8 is activated by death inducing signaling complex which then cleaves and activates caspase-3 and results in apoptotic cell death [
65]. Our results demonstrated that fungal EDT increased the levels of cleaved, PARP in a dose-dependent manner, indicating that intrinsic pathway was involved in the process of fungal EDT-induced apoptosis in HepG2 cells. The intrinsic apoptosis pathway is largely caspase 9-dependent which further activates caspase-3 which has PARP as a substrate, among others. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis [
66].
The increased Bax/Bcl-2 ratio leads to ΔΨm collapse, cytochrome c release, caspase-3 activation, and eventually apoptosis [
67]. In addition, pro-apoptotic protein Bax is closely associated with the control of mitochondrial membrane permeability and release of cytochrome c [
68]. Our results showed that fungal EDT increased the levels of Bax (Pro apoptotic protein) decreased the levels of Bcl-2 (an anti-apoptotic protein) and cleavage of PARP together with signalling factor p38 in a dose-dependent manner, confirming that fungal EDT-induced apoptosis in HepG2 cells. Mitogen-Activated Protein Kinases (MAPKs) have been described as the major oxidative stress-sensitive signal transducing pathways [
69] and serve as upstream signals for the initiation of apoptosis. JNK and p38 MAPK have been shown to get activated response to ROS generation and mitochondrial dysfunction, which are frequently associated with the induction of apoptosis [
70]. Previous studies have reported natural compounds induced apoptosis in HepG2 cells through ROS mediated MAPK activation and mitochondrial dysfunction [
71,
72]. Our results showed that fungal EDT significantly increased the level of p38 in a dose-dependent manner without affecting the expression of total proteins, indicating that these MAPK pathways were activated in the process of fungal EDT -induced apoptosis in HepG2 cells.
Acknowledgements
This work was supported by the University Grant Commission and DST-SERB (DST No: SERB/LS-412/2013 dated 20/09/2013) GOI, New Delhi, India. We also thank DBT-IISc Partnership programme. DST-FIST and UGC Special Assistance Programme for financial support and proving facilities. KS is thankful to the spectroscopy /analytical test facility and Sophisticated Instruments Facility (SIF) at the Indian Institute of Science, Bangalore for chromatography and spectroscopy analysis. KS gratefully acknowledges Dr. Karthikeyan Subburayan currently working at Department of Biology, New York University Abu Dhabi, United Arab Emirates for kindly providing PARP antibody and his support in cell imaging analysis. The authors thank Mr. Prashneel Goundar for critically reading the manuscript.