Current Cancer Drug Targets

Current Cancer Drug Targets

74

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Current Cancer Drug Targets, 2017, 17, 74-88

RESEARCH ARTICLE ISSN: 1568-0096 eISSN: 1873-5576

Strong Anti-tumorous Potential of Nardostachys jatamansi Rhizome Extract on Glioblastoma and In Silico Analysis of its Molecular Drug Targets

Impact Factor: 3.707

BENTHAM SCIENCE

Himanshi Kapoor1, Nalini Yadav2, Madhu Chopra2, Sushil Chandra Mahapatra3 and Veena Agrawal1,* 1

Medicinal Plant Biotechnology and Applied Research Laboratory, Department of Botany, University of Delhi, Delhi, India; 2Molecular Modelling and Anti-Cancer Drug Development Laboratory, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India; 3Department of Physiology, All India Institute of Medical Sciences, New Delhi, India Abstract: Background: Glioblastoma has been reckoned as the prime cause of death due to brain tumours, being the most invasive and lethal. Available treatment options, i.e. surgery, radiotherapy, chemotherapy and targeted therapies are not effective in improving prognosis, so an alternate therapy is insistent. Plant based drugs are efficient due to their synergistic action, multi-targeted approach and least side effects. Methods: The anti-tumorous potential of Nardostachys jatamansi rhizome extract (NJRE) on U87 MG cell line was evaluated through various in vitro and in silico bio-analytical tools. ARTICLE HISTORY Received: May 13, 2016 Revised: August 27, 2016 Accepted: September 28, 2016 DOI: 10.2174/157016381366616101914 3740

Results: NJRE had a strong anti-proliferative effect on U87 MG cells, Its IC50 was 33.73±3.5, 30.59±3.4 and 28.39±2.9 µg/mL, respectively after 24, 48 and 72 h. NJRE at 30 µg/mL induced DNA fragmentation, indicating apoptosis, early apoptosis began in the cells at 20 µg/mL, whereas higher doses exhibited late apoptosis as revealed by dual fluorescence staining. NJRE at 60 and 80 µg /mL caused a G0/G1 arrest and at 20 and 40 µg/mL showed excessive nucleation and mitotic catastrophe in the cells. Immuno-blotting validated the apoptotic mode of cell death through intrinsic pathway. NJRE was harmless to normal cells. In silico docking of NJRE marker compounds: oroselol, jatamansinol, nardostachysin, jatamansinone and nardosinone have revealed their synergistic and multi-targeted interactions with Vestigial endothelial growth factor receptor 2 (VEGFR2), Cyclin dependent kinase 2 (CDK2), B-cell lymphoma 2 (BCL2) and Epidermal growth factor receptor (EGFR). Conclusion: A strong dose specific and time dependent anti-tumorous potential of NJRE on U87 MG cells was seen. The extract can be used for the development of safe and multi-targeted therapy to manage glioblastoma, which has not been reported earlier.

Keywords: Glioblastoma, Nardostachys jatamansi, anti-proliferative effect, alternative medicine research, molecular docking, pharmacokinetics. INTRODUCTION Every year, 240,000 cases of brain and nervous system tumors are reported worldwide out of which Glioblastoma multiforme, a high grade (IV) tumor of the astrocytes, is the most common (accounting 80 % of all malignant brain cancers) with high incidence and mortality rates. Headaches, nausea, seizures, cognitive and personality changes and paralysis are some of the symptoms of the disease which makes survival extremely difficult, ultimately leading to death after 12-14 months of diagnosis. The available management options (depending upon the location of the tumor, health and age of the patient) include radiation as the mainstay, supported by surgery and chemotherapy and more recently targeted biological therapies. Despite, multimodal treatment, the median survival rate of the patients remains extremely low attributed to its highly invasive nature which causes *Address correspondence to this author at the Medicinal Plant Biotechnology and Applied Research Laboratory, Department of Botany, University of Delhi, Delhi, India; Tel: +91 112766 6802; E-mail: [email protected] 1873-5576/17 $58.00+.00

quick deterioration [1-8]. Incidentally, a slight increase in the median survival beyond 12 months (up-to a maximum of 15 months) was seen when this was treated with temozolomide and radiation [9]. Therefore, there is an extreme exigency for search of an alternate therapy for the treatment of glioblastoma. Plant based drugs play an important role in the treatment of cancers; vinca alkaloids (vinblastine, vincristine), taxol, etiposide, teniposide [10], etc. have reached to commercial scale. Herbal drugs are preferred because they have least side effects and are highly effective due to synergistic and multi-targeted action [11-15]. Nardostachys jatamansi (D.Don) DC. a promising medicinal herb of the high altitude has been used to treat a number of brain and memory related disorders [16-18]. However, it has not been used for treating glioblastoma. The present investigation for the first time reports strong efficacy of N. jatamansi rhizome extract (NJRE) on U87 MG glioblastoma cell line along with molecular docking studies of its five marker compounds (oroselol, jatamansinol, nardostachysin, jatamansinone and nardosinone) with four important drug targets for cancer: Vestigial endothelial growth factor receptor 2 (VEGFR2), Cyclin dependent kinase 2 (CDK2), B-cell lymphoma 2 © 2017 Bentham Science Publishers

Strong Anti-tumorous Potential of Nardostachys jatamansi

(BCL2), and Epidermal growth factor receptor (EGFR). Computational tools such as docking have paved the way for studying molecular interactions and simulations of the drug molecules with their targets on a broad scale basis [19]. In silico studies in the current investigation have highlighted a probable multi-targeted therapeutic approach of NJRE compounds in cancer, not done earlier. MATERIALS AND METHODS Nardostachys jatamansi Rhizome Extract (NJRE) Rhizomes of N. jatamansi (Department of Botany, University of Delhi, Accession no. 14244) were harvested from the dried plants obtained from Uttarakhand, India. These were extracted in methanol (48 h) and evaporated to dryness. One milligram of this extract was dissolved in 10 µ L of DMSO; further 990 µL of incomplete media of the respective cell line was added to obtain NJRE stock. Various dilutions of this stock were used for experiments. Cell Lines and Culture U87 MG (Astrocytoma-Glioblastoma), normal cell line HEK (Human Embryonic Kidney) and other cell lines {U373 MG (Astrocytoma-Glioblastoma), Mia-PaCa-2 (Pancreatic cancer), Colo-320 DM (Colon cancer) and Hep-3B (Liver cancer)} were procured and authenticated from National Centre for Cell Sciences, India. These were grown in their respective media (Sigma, USA) supplemented with 10% fetal calf serum (Gibco, USA) and 1% antibioticantimycotic solution (Hi-Media, India). The cells (70-80% confluency) less than five passages were used for experiments. The cells were maintained in a humidified CO2 (5%) incubator (Shell Lab, USA) at 37 °C. Effect of NJRE on Cell Viability (U87 MG) Cells (5000/ well) were seeded (24 h) in 96 well plates for MTT {3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide} assay [20]. Cells were treated with NJRE (10-60 µg/mL) and positive control [methotrexate (0.25, 5, 10, 20, 40 and 80 µg/mL)], keeping alongside, vehicle control (0.1% DMSO in the respective incomplete media of the cell line) and control (all in triplicates) for 24, 48 and 72 h at 37 °C. Twenty microlitres of MTT (Sigma, USA) reagent (5 mg/mL) was added to each well, followed by incubation for 4 h, subsequently MTT was removed and 150 µL of DMSO was added. The plates were read (Tecan, Austria) at 540 nm (reference: 630 nm).

Current Cancer Drug Targets, 2017, Vol. 17, No. 1

75

quently, these were treated with different NJRE concentrations (20, 40, 60 and 80 µg/mL), keeping the controls alongside for 24, 48 and 72 h and viewed under an inverted microscope (Nikon, Japan. The photographic data was recorded. DNA Fragmentation Cells (0.1x106) were cultured (24 h) in each 40 mm petridish. These cells were exposed to different NJRE concentrations (10-70 µg/mL) for 24 h. The monolayer of cells from the treated and control groups was harvested for DNA isolation [21] which was run on a 1.5% agarose (Sigma, USA) gel, at 90 V for 30 min. Dual Fluorescence Staining by Acridine Orange and Ethidium Bromide (AO/EB) Cells (0.1x106) were seeded (24 h) in 40 mm petri-dishes and treated with given NJRE concentrations for 24, 48 and 72 h. Subsequently, these cells were harvested and washed with 100 µ L PBS thrice. The final pellet was dissolved in PBS (25 µL), 2 µL AO/EB (Sigma, USA) (100 µg/mL) dye mix was added to it, incubated (10 min) and viewed under fluorescence microscope (Carl Zeiss, Germany) [FITC (502526 nm) and TRed (510-595 nm) filters] at 10 and 100× magnifications [22]. Assessment of Mitotic Catastrophe by Dapi Staining Cells of each treatment and controls were treated with hypotonic 1% Sodium citrate solution and incubated (5min) for DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining [23]. Such cells were fixed in methanol: acetic acid (3:1) solution, air dried on a clean glass slide, stained with DAPI (Sigma, USA) (1µg/mL) and viewed at 10 and 100× magnifications under fluorescence microscope [DAPI filter (350-470 nm)]. Such slides were screened for: micronuclei, nuclear budding and chromatin condensation. Comet Assay (Single Cell Gel Electrophoresis) For comet assay the modified protocol [24] was followed. The slides were processed and analyzed under fluorescence microscope [TRed filter, magnification: 10, 20 and 40×] and Olive Tail Moment (OTM) was calculated. OTM= %Tail DNA × [Centre of Mass (Head)-Centre of Mass (Tail)]/100 Cell Cycle Analysis

Trypan Blue Exclusion U87 MG cells (0.1x106) were seeded (24 h) and treated with the given NJRE concentrations for 24, 48 and 72 h. The cells from the treated and control groups were stained with trypan blue (Sigma, USA) dye (4%) in 1:1 ratio. The dead and live cells were counted under a light microscope (Carl Zeiss, Germany) using a haemocytometer.

In each 4 mm petri-dishes, 0.1x106 cells were seeded (in serum free media for cell phase synchronization) (24 h). Both control and treated cells were processed for cell cycle analysis using the referred protocol [25] and analyzed using a flow cytometer (BD Accuri C6, USA) equipped with BD Accuri C6 software for gating and analysis. Clonogenic Assay

Morphology Analysis For observing the cellular morphology, 0.1x106 cells were seeded in each 40 mm petri-dishes for 24 h. Subse-

Clonogenic assay was performed using the modified protocol [26]. The cells of each treatment and controls were harvested, counted and 50 cells per petri-dish were seeded

76 Current Cancer Drug Targets, 2017, Vol. 17, No. 1

for each sample and incubated for a week. The colonies were stained with crystal violet (1%) and counted using a stereomicroscope (Carl Zeiss, Axioscope, Germany). The Effect of NJRE on the Normal Cell Line HEK and Other Cell Lines The effect of NJRE on the normal cell line HEK and other cell lines such as U373 MG, Mia-paca-2, Colo-320 DM and Hep-3B was determined through MTT as described above. Immuno-blotting U87 MG cells were lysed in RIPA buffer (Sigma, USA) after 24h of treatment. The protein concentration was estimated by Bradford Assay (in triplicate) using bovine serum albumin (BSA) as standard. The proteins were electrophoresed on a 12% SDS-polyacrylamide gel (the concentration of protein per well was 20-30 µg) followed by transfer on a nitrocellulose membrane (Bio-Rad Laboratories, Berkely, USA). The membrane was blocked in 5% BSA in tris buffer saline and Tween 20 pH 7.5 (TBST) overnight to prevent non-specific binding. It was then incubated in primary antibody [caspase 3, caspase 9, poly ADP ribose polymerase (PARP) and β -actin (Cell Signalling technology, Beverly, MA, USA) diluted (1 : 1000) in 5% BSA in TBST)] for 2 h at RT. Subsequently, the membrane was washed thrice with TBST for 5 min each and incubated with horseradish peroxidase conjugated secondary antibody [anti-rabbit, anti-mouse and anti-goat (Cell Signalling technology, Beverly, MA, USA) diluted (1 : 2000) in 5% BSA in TBST)] for 1 h at RT. The membrane was washed thrice with TBST and developed with luminol reagent (Bio-Rad) following the manufacturer's protocol and chemiluminescence was recorded (Bio-Rad, Chem-doc) using Image Lab software. ADME Properties The ADME properties of the NJRE marker compounds: Oroselol, Jatamansinol, Nardostachysin, Jatamansinone and Nardosinone (PubChem CID: 160600, 759302, 10598736, 759294 and 168136, respectively) were determined using QikProp (Schrodinger, USA) [27]. Out of many pharmacokinetic parameters available in the software, relevant ones were enumerated. Further, the drug likeliness of the compounds was checked on the basis of Lipinski’s rule of five [28] and #stars descriptor given in the prediction. Docking Docking was performed in the Discovery Studio (DS) Client v 4 package (Accelrys Inc., CA, USA). The X-ray crystal structures of the receptors: VEGFR2, EGFR, CDK2, BCL2 were obtained from the RCSB protein data bank, PDB ID: 5EW3, 4V0G, 4D1X, and 4LVT, respectively. The structures of the above mentioned marker compounds were constructed in DS. Both the receptors and compounds were prepared for docking by removing water, adding Hydrogen atoms, valency monitoring, cleaning geometry and applying CHARMm force field. The energy minimization of the ligand was performed using the steepest descent and conjugate gradient algorithm. The site sphere (active site) of the

Kapoor et al.

receptor was kept same as of the original ligand. Docking simulations were performed by the CDOCKER program which generates ten random conformations of ligands within the active site through high-temperature molecular dynamics. The receptor-ligand complex was ranked based on CDOCKER interaction energy (CDI) in kcal/mol and LUDI score values [29]. Statistical Analysis All the experiments were performed three times and subjected to two-way ANOVA (post hoc: tukey test, P < 0.05), also correlation and regression analysis were performed. The results were presented as mean±SD. RESULTS Effect of NJRE on Cell Viability (U87 MG) Cell viability was evaluated through MTT assay. In MTT assay, yellow coloured aqueous solution of MTT [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], gets reduced by the mitochondrial dehydrogenases of metabolically active cells to produce purple formazan crystals, which is spectrophotometrically determined. The absorbance thus obtained is directly proportional to the number of viable cells. U87 MG cells when treated with 10, 20, 30, 40, 50 and 60 µg/mL concentrations of NJRE for 24, 48 and 72h. A dose and time dependent decrease in cell viability was recorded. The IC50, as determined by sigmoidal curve (Graph Prism 6, CA) was 33.73±3.5 (r=−0.96, R2= 0.95), 30.59±3.4 (r=−0.96, R2= 0.94), and 28.38±2.9 µg/mL (r=−0.95, R2 = 0.92) for 24, 48 and 72 h, respectively. The above said results indicated that the effect of NJRE concentrations was more significant than time duration on the cell viability. Furthermore, the viability and concentration correlated negatively and represented a good linear fit (R2 ~1) of the model (Fig. 1A). Vehicle control (VC) had the cell viability percent similar to the control, whereas the positive control (methotrexate) had less cell viability (Fig. 1A). Trypan Blue Exclusion Cell viability of U87 MG cells after NJRE treatment was also assessed by trypan blue exclusion. The number of live cells after NJRE treatment, decreased sharply, with the increasing drug concentrations and time duration {R2= 0.91, r=−0.96 (24 h), R2= 0.96, r=−0.98 (48 h), R2=0.96, r=−0.98 (72 h)}. At 40 µg/mL, the viable cell counts approximately reduced to half compared to control. However, both the control and VC exhibited similar viable cell counts. Interestingly, the cell counts of higher doses (60 and 80 µg/mL) were similar to that of methotrexate (50 µg/mL) (Fig. 1B). Morphology Analysis Since NJRE caused an inhibition of U87 MG cells, its effects on the cellular morphology were studied further. At 20 µg/mL, morphology of cells was similar to that of control and VC, except for shape distortion, and increased number of dead cells. At 40 µg/mL, hallmark of apoptosis, i.e. cell shrinkage, membrane blebbing and echinoid processes were observed with concomitant increase in number of dead cells.



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Fig. (1). A: Sigmoidal curve of NJRE dose response. B: Trypan Blue Exclusion:- Live cells (x106)/mL cell suspension versus samples plot. C: AO/EB:- Percent apoptotic cells versus samples plot. D: Comet assay:- OTM (µm) versus samples plot. E: DNA fragmentation [1 and 9: Marker, 2: Control, 3: Vehicle control, 4-8: 10-50 µg/mL NJRE, 10-11: 60-70 µg/mL NJRE and 12-16: 10-50 µg/mL methotrexate, respectively. F-L: Effect of different NJRE doses on morphology of U87 MG cells after 72h, Order: control, vehicle control, positive control, 20, 40, 60 and 80 µg/mL, respectively. Scale: bar=2 µm, Magnification: 40×. Arrows: dead cells and apoptotic features such as echinoid processes, cell shrinkage and nuclear condensation. Letters on the top of each column indicate significant differences according to Duncan’s multiple range test at p = 0.05. (Abbreviations: C: Control, VC: Vehicle control, PC: Positive control and 20, 40, 60 and 80 are different doses of NJRE in µg/mL).

At 60 and 80 µ g/mL cell morphology was completely distorted, culminating in rounded dead apoptotic cells, similar to those of methotrexate (50 µg/mL). Detrimental effect of

both increasing drug concentrations and time duration on the cell morphology has been clearly depicted (Fig. 1F-L, S1).


DNA Fragmentation To further confirm that NJRE caused apoptosis, DNA was isolated from the NJRE treated cells. A distinctive ladder like pattern of the cleaved DNA, was visible on the gel at 30 µg/mL and above concentrations indicating apoptosis (Fig. 1E). The DNA of the control and VC was intact whereas those treated with methotrexate also exhibited DNA fragmentation. Dual Fluorescence Staining by AO/EB Acridine orange and ethidium bromide (AO/EB) dual fluorescence staining of NJRE treated cells again pointed to the apoptotic mode of cell death and loss of membrane integrity. At 60 and 80 µg/mL, reddish fragmented nuclei were observed, with percent apoptotic cells: (87.5±1.8, 99.4±0.98), (89.49±4.12, 100±0.0) and (95.57±6.49, 100±0.0) for 24, 48 and 72 h, respectively. At 40 µg/mL, apoptotic cell percent was 55.53±8.8 at 24, 61.89±3.5 at 48 and 63.8±2.49 at 72 h. A less number of apoptotic cells were seen at 20 µg/mL. The increase in apoptotic cells with increasing drug concentrations and time duration generated a good linear fit of the model {R2= 0.98, r=0.96 (24 h), R2 = 0.98, r=0.99 (48 h), R2=0.96, r= 0.98 (72 h)} (Figs. 1C and 2A-G, S2). The cells of control and VC fluoresced green and did not show apoptosis, as ethidium bromide will only cross the cells with damaged membranes to intercalate nuclear DNA and such cells emitted red fluorescence. Such type of studies have been reported for the first time in the case of NJRE. Assessment of Mitotic Catastrophe by DAPI Staining DAPI stained DNA (live and fixed cells) produces blue fluorescence under ultraviolet light. The normal cells (control and VC) appeared round and fluoresced blue with clear margin whereas the apoptotic cells showed abnormal margin and condensed nuclei (Figs. 2H-N). Two nuclear conditions were observed in cells after NJRE treatment: (i) pyknosis manifested as nuclear condensation and shrinkage, (ii) yorrhexis exhibited as nuclear fragmentation. Twenty and 40 µg/mL showed pyknosis and micronuclei formation, thereby indicating mitotic catastrophe in U87 MG cells. Incidentally, positive control and 60 µg/mL exhibited both pyknosis and yorrhexis, whereas at 80 µg/mL only yorrhexis was seen. (Fig. 2H-N, S3). No such report of NJRE causing mitotic catastrophe in other cell lines or other plant extracts causing mitotic catastrophe in U87 MG cell line is available. Comet Assay DNA damage was assessed by alkaline comet assay (single cell gel electrophoresis) for the detection of both single and double stranded DNA breaks and alkali-labile DNA adducts. An increase in DNA damage was observed with increasing NJRE concentrations as assessed by increasing percentages of the tail DNA vis-a-vis decreasing head DNA and increasing Olive Tail Moment (OTM), which is a measure of tail DNA moment from the nucleus. More the OTM, more will be the DNA damage and hence, more inhibition of DNA replication. OTM was highest at 80 µg/mL, i.e. 66.29±0.76, 97.62±4.67 and 143.82±4.24 µm for 24, 48 and 72 h, respec-

Kapoor et al.

tively. A strong positive correlation of OTM was observed with the increasing drug and time concentrations {r=0.98(24 h), r=0.98(48 h), r=0.97(72 h). Regression coefficient R2 {R2=0.97(24 h), R2=0.96(48 h), R2=0.95(72 h)} indicated a good fit of the linear model. Control and VC had least OTM (Figs. 1D and 2O-U, S4). This is also being reported for the first time in the case of NJRE. Cell Cycle Analysis Higher doses (60 and 80 µg/mL) of NJRE caused cell cycle arrest at G0/G1 phase in U87 MG cells as determined through flow cytometric cell cycle analysis after propidium iodide staining (Figs. 3C and D). Methotrexate (50 µg/mL) also caused a G0/G1 arrest whereas the control and VC exhibited normal cell cycle phases. Results were statistically significant and generated a good fit of the linear model (R2=0.86 and r=0.91). Clonogenic Assay Clonogenic assay is a well known standard procedure to determine the anti-proliferative effect of the drugs. With an increase in NJRE concentrations, there was a decrease in cell proliferation (R2=0.966, r=−0.96) (Figs. 3A and B). Average number of colonies were 58±3, 60.6±2.08, 2±1, 56.3±2.5, 23±1, 18.3±0.57 and 2.3±1.52, respectively for control, VC, positive control, 20, 40, 60 and 80 µg/mL. The results indicated that, NJRE reduced the clonogenic capacity of U87 MG cells, thereby indicating its probable role in the reduction of metastasis. The Effect of NJRE on Normal Cell Line HEK and Other Cell Lines No cyto-toxic effects of NJRE on HEK were seen. At its different doses (10, 20, 30, 40, 50 and 60 µg/mL), the percent viability values after 24 h of treatment were: 83±3.6, 80±2.5, 75.7±3.5, 75.8±2.4, 76.3±2.5 and 77.3±4.1, respectively (Fig. S5). The R2 and r values were 0.5 and −0.7, respectively, thus, indicating poor fit of the linear model. Besides, NJRE exhibited dose dependent inhibition on other cell lines such as U373 MG, Colo-320 DM, Mia-PaCa-2 and Hep3B. The best response was seen on U373 MG cell line, followed by Colo-320DM, Hep-3B and Mia-PaCa-2 (Fig. 3E). NJRE Induces Apoptosis By Intrinsic Pathway in the U87 MG Cells NJRE was able to induce cell death by apoptosis in the U87 MG cells as validated by immuno-blotting. There was a decrease in the expression of caspase 3, caspase 9 and PARP in the treated cells and positive control versus control and vehicle control, thereby indicating the activation of mitochondrial mediated intrinsic pathway of apoptosis (Fig. 4). Pharmacokinetics of the Marker Compounds of Nardostachys jatamansi Pharmacokinetic profiles of the marker compounds: oroselol, jatamansinol, nardostachysin, jatamansinone, nardosinone studied by Qik Prop [27] revealed a strong drug likeliness of the compounds in accordance with the Lipinski's rule of five (neither of the compounds violated any



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Fig. (2). Effect of NJRE on U87 MG (72h) A-G: AO/EB staining for apoptosis [Arrows: dead cells and apoptotic features such as nuclear condensation and fragmentation and apoptotic bodies], H-N: DAPI staining for mitotic catastrophe [Red arrows: nuclear condensation and fragmentation and yellow arrows: micronuclei and nuclear budding] and O-U: Comet assay for DNA damage. Order: control (A, H, O), vehicle control (B, I, P), positive control (C, J, Q), 20 (D, K, R), 40 (E, L1, L2, S), 60 (F, M1, M2, T), and 80 µg/mL (G, N, U) NJRE, respectively. Scale: bar=20 µm, Magnification: 100× for A-N, Scale: bar=50 µm, Magnification: 40× for O-U.


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Fig. (3). A: Anti-proliferative effect of NJRE on U87 MG cell line as seen by Clonogenic assay: a: control, b: vehicle control, c: positive control, d-g: 20, 40, 60 and 80 µg/mL NJRE, respectively. B: Clonogenic assay:- Number of colonies versus samples plot C: Cell cycle peaks a: control, b: vehicle control, c: positive control, d-g: 20, 40, 60 and 80 µg/mL NJRE. D: Graph of cell cycle analysis. E: Radar plot: effect of NJRE on other cancer cell lines. Letters on the top of each column indicate significant differences according to Duncan’s multiple range test at p = 0.05, (Abbreviations: C: Control, VC: Vehicle control, PC: Positive control and 20, 40, 60 and 80 are different doses of NJRE in µg/mL).



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ADME properties of the Marker compounds of Nardostachys jatamansi.

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-0.424

444.649

-0.17

-2.973

3

0

Jatamansinone

0

825.895

-0.418

402.305

-0.497

-2.365

3

0

Oroselol

0

837.85

-0.544

408.603

-0.018

-3.276

3

0

Nardosinone

0

1689.974

-0.033

872.291

-0.21

-2.285

3

0

Nardostachysin

-1

693.464

-0.966

333.055

0.78

-5.827

3

0

QPPMDCK(500 great) 1.5 to 1.5)

Lipinski's rule) and other important parameters such as Molecular weight, Hydrogen bond donors, Hydrogen bond acceptors, Predicted octanol/water partition coefficient (QPlogPo/w), Rotable bonds, Van der Waals surface area of polar nitrogen and oxygen atoms (PSA), Total solvent accessible surface area (SASA), Prediction of binding to human serum albumin (QPlogKhsa), Predicted aqueous solubility, log S (QPlogS) and Human oral absorption (Table 1). Since, NJRE is being used for glioblastoma, the CNS (Central Nervous System) activity and Blood brain barrier crossing ability of its marker compounds were also evaluated. Incidentally these compounds exhibited a moderate CNS activity and a high blood brain barrier crossing ability. The blood barrier penetration ability was assessed in terms of properties such as Predicted apparent Caco-2 cell permeability in nm/sec (QPPCaco), Caco-2 cells are a model for the gutblood barrier, Predicted brain/blood partition coefficient (QPlogBB) and Predicted apparent MDCK cell permeability in nm/sec (QPPMDCK), MDCK cells are considered to be a good mimic for the blood-brain barrier. Drugs targeted for

CNS should have low PSA (Polar surface area) between 6070 A [30], and the marker compounds had PSA in this range, except for nardostachysin. Apart from these #stars descriptor (number of property or descriptor values that fall outside the 95% range of similar values for known drugs) was also determined, the compounds had zero stars, except for nardosinone which had 1 star, a large number of stars suggests that a molecule is less drug-like than molecules with few stars Table 1. Docking The docking studies performed for the marker compounds, on four protein receptors revealed a differential receptor-ligand interaction in each case as assessed by CDOCKER interaction energy and ludi score values (Tables 2, 3, Fig. 5). The RMSD (Root Mean Square Deviation) values of the control ligands with the receptors were 2 or less than 2 for each case. The interactions (based on CDOCKER interaction energy and ludi score values) of the marker


Table 2.

Kapoor et al.

The Receptor- ligand energies [CDOCKER Interaction Energy (CDI) in kcal/mol] and scores after docking. VEGFR2

BCL2

EGFR

CDK2

Compounds CDI

Ludi Score

CDI

Ludi Score

CDI

Ludi Score

CDI

Ludi Score

Jatamansinol

-30.8929

650

-24.4657

481

-26.7738

166

-34.0533

403

Jatamansinone

-17.058

622

-23.9538

543

-27.5095

165

-34.466

357

Oroselol

-31.153

612

-26.6042

553

-25.9156

192

-35.1496

466

Nardosinone

-18.969

475

-26.2517

357

-23.9302

139

-30.7755

347

Nardostachysin

-34.0731

529

-42.4247

372

-42.7983

172

-53.5325

554

Table 3.

The molecular interactions of the Marker compounds with the amino acids of the drug targets (VDW: Van der waals, E: Electrostatic, HB: Hydrogen bond, π: Pi, σ: Sigma). VEGFR2

CDK2

BCL2

EGFR

Compounds VDW

E

HB

Jatamansinol

E, D, C, A, V, E, L , F, L, G, F K

D

Jatamansinone

V, A, L, C, D, K, F, G, E L

-

π

VDW

E

HB

VDW

E

HB

F,C, I, L, K, G, E, Z N, D, L V

Q

Y, V, R, F, G, F, N D,A

A

F

T, K, N, G, E, K, D, E, V, D, F, H, F, Q A, Q, L, I

K

R, N, A, V, Y, R, D, F, Y G

Y

Oroselol

I, L, R, C, V

E, H, I

H

-

A, V, N, F, E, N, V, F, A, D, K, T K (two) R, G L, Q, I, K, G D, Y

G

Nardosinone

L, V, F, I, A

K, E, C, D

-

-

K, L, D, V, G, D, I, K, T, N, Q E

-

-

L, E, W, V, V, E, I, L, F, E, Q, N, Y, G, R (two), T (two), F, A, Y, D, D, G, H, Q, K, G, T, N, R N, G K, Q R A L,D

C, H, I, L, K, E, E, D Nardostachysin V, R, F, G, D E, A

compounds with the receptors were in the order VEGFR2> CDK2> BCL2> EGFR. Out of the five marker compounds, oroselol docked favourably to all the receptors followed by jatamansinol, nardostachysin, jatamansinone and nardosinone (Tables 2, 3 and Figs. 5, 6A). DISCUSSION Natural products have opened up new vistas in management of cancer as has been proved in several existing products [31]. The probable reason is attributed to their synergistic action and multi-targeted approach with minimum side effects [10-14]. Present investigation for the first time reports the strong anti-cancerous activity of the rhizome extract of Nardostachys jatamansi on U87 MG human glioblastoma cell line. Though some fragmentary reports on the in vitro cyto-toxicity of rhizome extract of N. jatamansi on other cancer cells are available, but none of these pertains to glioblastoma. In vitro cyto-toxicity of N. jatamansi root extract against human neuroblastoma cancer cell lines: IMR-32 and SK-N-SH was reported [32] wherein its 95% alcoholic extract at 100 µg/mL exhibited 71% and 85% cell death, respectively. Furthermore, in yet another case, cyto-toxic activity of the alcoholic extract of N. jatamansi rhizomes and its n-butanol fraction on human lung (A549), liver (Hep-2), prostate (PC-3) and ovarian (OVCAR–5) cancer cell lines after 48 h of incubation was also seen, but IC50 value was not

K

L, W, G, F, A, D, R, V, Y Y

π

σ

Y (two) G

F, R

-

F (two), R

VDW

E

HB π σ

Y, L, G, A, G, V, S K

V

G G

N, A, V, G, D, K, L, M, Y L, E

L

- -

S, G, L, P, K, V, V A, E, Y G, C,L

L G

-

- -

C

- -

-

-

M, A, Y, L, C, D, V, G, N, R

-

-

D, G, N, L, R, C, V, A, M, E, L Y

-

mentioned [33]. Recently, the anti-cancerous activity of the methanolic extract of N. jatamansi rhizomes on breast cancer cell lines MCF-7 and MDA-MB-231 was evaluated [34] with their IC50 values being 60±4.78 and 23.83±0.69 µg/mL, respectively, after 48 h of incubation. Surprisingly, in the present case, NJRE proved most effective on U87 MG cell line even at minimal doses in causing lethality, its IC50 value being 33.73±3.5, 30.59±3.4 and 28.38±2.9 µg/mL for 24, 48 and 72 h, respectively. In vivo anti-tumorous activity of N. jatamansi extract was evaluated against Sarcoma-180 solid tumor model at 100 and 200 mg/kg body weight and a significant 29.53 percent tumor growth inhibition was observed in the latter [33]. Referring to the efficacy of other plant extracts on U87 MG cell line, mention can be made of the anti-proliferative effect of six Salvia species of which only Salvia menthaefolia induced cyto-toxicity in the cells with its much higher IC50 value being 238 µg/mL after 72 h of treatment [35]. In another case, the anti-tumor activity of Pterodon emarginatus fruit extract on U87 MG cell line revealed a very high IC50 value, i.e. 10 mg/mL, after 48 and 72 h of incubation [36]. More recently, the IC50 value for in vitro cyto-toxic activity of the isolates of Randia dumetorum bark on U87 MG was seen to be 166 µg/mL [37]. Contrary to the above reports, in the present investigation, remarkable cyto-toxic effects of N. jatamansi rhizome extract (both dose and time



A1

A VAL999 GLU917 VAL916 PHE918 TRS919

LEU1035

LEU A:840

CYS1045

PMF

GLU885

GL

A:1317

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886

PHF1047

GLU A:885

LYS968 LEU840

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AL145

ALA97

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TYR105

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van der Waals ARG A: 104

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H

Pi

Electrostatic

Metal

ASP A: 100

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Residue Interaction

Covalent bond

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TYR A: 199

83


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Kapoor et al.

C1

PRO806 GLY906

LEU828

LEU A:905

CYS909

GLY829

Signa

LEU905 LEU956

LYS310 ASN85404

GLY831 SER835

G LU A:903

VAL835 AU 853

ALA966 GLU903

TYR A:904

P RO A:906

G LY A:829

ASP867 LYS855

G LY A:908

ALA A:853

C YS A:909

Sigma

MET902

VA L A:836

Residue Interaction

LYS A:830

Electrostatic van der Waals

O H

LEU A:956

O

Pi

O

O

SER A:835

LEU A:828

Covalent bond Water

G LY A:831

LYS89

Metal

GUN85 LEU134

ASP86

IIS84 LEU03

VAL11 PHE82 GUL81

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PHE80

ALA31

G LU A:12 G LY A:11

ASN A:132

O

H GLN A:131

O H

G LY A:13

LYS A:89 HIS A:84

O O THR A:14

Residue Interaction Electrostatic van der Waals Covalent bond

O

O

LYS VA L A:129 A:18

ASP A:145

LEU A:134 PHE A:82

ALA A:31

Water Metal

ASP A:86

ILE A:10

LYS A:33

ALA A:144 PHE A:80

GLN A:85

VA L A:64

LEU A:83

G LU A:82

Fig. (5). Molecular interactions of the NJRE marker compounds with different drug targets as shown by 2D and 3D diagrams of In silico docking: A-A1: Jatamansinol-VEGFR2, B-B1: Oroselol-BCL2, C-C1: Oroselol-EGFR, D-D1: Nardostachysin-CDK2.



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Fig. (6). A: The comparative interactions of NJRE Marker compounds with different drug targets B: Multi-targeted and synergistic action NJRE marker compounds as revealed by in silico studies.

kinetics) on U87 MG cell line were observed. Its IC50 value was the lowest compared to all the above reports, i.e. 33.73±3.5 µg/mL at the minimum incubation (24 h), which further decreased to 30.59±3.4 µg/mL when the treatment was increased to 48 h, and it was the least, i.e. 28.38±2.9 µg/mL after 72 h of treatment. The low IC50 value of NJRE on U87 MG cells evidently proved its strong cyto-toxic and anti-proliferative potential, compared to the plant extracts referred earlier against the same cell line. Methotrexate was used as the positive control in the entire study. For MTT its different doses were tried, however for other experiments, 50 µg/mL was used (as selected through MTT). Likewise methotrexate, NJRE also caused cell death in the U87 MG cells but its mechanism of action

was different, i.e. it was cytocidal in nature, whereas methotrexate was cyto-static. All the results obtained were statistically significant as determined by tukey test. The results of both DAPI staining and cell cycle analysis pointed that NJRE caused mitotic catastrophe at lower doses 20 and 40 µg/mL, whereas it led to cell cycle arrest at the higher doses such as 60 and 80 µg/mL. A G2/M and G0/G1 phase arrest in breast cancer cell lines has been reported with N. jatamansi extract and its fractions [34], but not against glioblastoma. In a similar study, N. jatamansi extract and fractions have been known to reduce the clonogenic capacity of breast cancer cell lines [34]. NJRE induced apoptosis in the U87 MG cells as the hallmarks of apoptosis such as membrane blebbing, echinoid processes, pyknosis and yor-


rhexis were clearly visible. Immuno-blotting assay further validated the apoptotic mode of cell death through intrinsic pathway as the expressions of caspase 3, caspase 9 and PARP showed the expected declining trend in the treated cells and positive control compared to control and vehicle control. Needless to say, NJRE has shown very minimal toxic effects on normal cells (HEK). The methanolic extract of N. jatamansi has an in-vitro cyto-protective effect against hydrogen peroxide induced oxidative damage in C6 glioma cell line [38], which is a normal cell line for glial cells. Furthermore, N. jatamansi rhizome extract has minimal neurotoxicity in rats [16]. Thus, the study is giving further insight for the development of a potent harmless drug for glioblastoma in near future. Apart from U87 MG cell line, NJRE also exhibited antiproliferative activity on other cancer cell lines such as U373 MG, Colo-320 DM, Mia-paca-2 and Hep-3B, as revealed in the present investigation. The U373 MG cell line was most susceptible, whereas the Mia-PaCa-2 cell line was least susceptible. The differential susceptibility of the cell lines may be due to different drug targets which interacted with NJRE and induced different signalling pathways in each case. In recent times, the in silico ADME analysis has been proved to be very useful tool in predicting the probable efficacy of many natural and synthetic compounds as drugs [39, 40]. The ADME analysis of the marker compounds revealed their probable drug likeliness, moderate CNS activity and a high blood brain barrier crossing ability, thus, making them suitable to be used against glioblastoma, as a vast majority of drugs including monoclonal antibodies are not able to cross an intact blood brain barrier. Such type of analysis using this software is being reported for the first time in the case of N. jatamansi. Another notable feature of the work is computer aided screening and molecular docking which are being proved as powerful tools in modern drug discovery. Molecular docking studies of the aforesaid marker compounds were performed with Vestigial Endothelial Growth Factor Receptor 2 (VEGFR2), Cyclin Dependent Kinase 2 (CDK2), B-Cell Lymphoma 2 (BCL2) and Epidermal Growth Factor Receptor (EGFR). Differential affinities of the compounds were seen for each receptor based on their structures. The interactions were mainly hydrophobic and electrostatic involving hydrogen bonds, pie bonds and sigma bonds (Table 3). Oroselol docked to all the receptors with low CDI and high ludi scores, exhibiting the best interactions with all the receptors, followed by jatamansinol, nardostachysin, jatamansinone and nardosinone. Oroselol, jatamansinol and nardostachysin have an OH moiety in their structures, this may be the reason for their lower interaction energies and high ludi scores as compared to jatamansinone and nardosinone, both with a prominent ketonic group. As already known, VEGFR2 is responsible for angiogenesis, invasion, metastasis, proliferation and survival in glioblastoma, its increased expression is responsible for transition from low grade to high grade glioblastoma. Since, glioblastoma is most angiogenic of the cancers, its survival, invasion and proliferation is deeply dependent on blood supply, therefore, VEGFR2 is an important target for its therapy [41-43]. All the marker compounds docked favourably to

Kapoor et al.

VEGFR2, thereby indicating their probable efficacy in controlling angiogenesis. EGFR is another receptor which regulates various processes in the cell such as cell proliferation, survival, differentiation and apoptosis. Mutational changes leading to over-expression of EGFR is a reason for the development of many malignancies including glioblastoma [43-46]. The marker compounds exhibited in silico interactions with EGFR, which may probably effect proliferation and survival of malignant cells. Cyclin dependent kinases (CDKs) are important for cell cycle regulation and progression, in cancer, these CDKs are over expressed and there occurs uncontrolled cell division [47, 48]. The marker compounds also target CDK2, thereby probably causing a cell cycle arrest, as experimentally validated by flow cytometry. BCL2 family of proteins have an anti-apoptotic nature. It is a target for therapy in cancer, because its increased expression leads to inhibition of apoptosis in tumor cells [49, 50]. The docking simulations of the marker compounds with BCL2 indicated that NJRE probably caused apoptosis in the cancer cells by inhibiting BCL2. Thus, NJRE exhibits a probable multi-targeted therapeutic effect on cancer cells, by potentially targeting processes like: angiogenesis, metastasis, proliferation and invasion. The multi-targeted effect is most likely due to the differential response of marker compounds with each receptors, hence, a synergistic action of healing is proposed (Fig. 6B), which is a well known characteristic of plant based medicine. With its marker compounds showing drug-like properties it is a safer alternative option to develop drug against Glioblastoma, finding a suitable therapy for which is required. Various other reports of computer guided approaches in drug discovery from natural products are available [51-56], but it has not been explored in the case of N. jatamansi for glioblastoma. Summarizing, strong anti-proliferative (dose specific and time dependent) effect of NJRE on U87 MG and other cell lines has been reported. It caused DNA damage, apoptosis, mitotic catastrophe (20 and 40 µg/mL) and cell cycle arrest (60 and 80 µg/mL) in the cells. It caused cell death due to intrinsic pathway of apoptosis, which is a novel finding of this investigation. With its marker compounds showing druglike properties, NJRE is a safer alternative to develop drugs against glioblastoma. Though innumerable papers have appeared on U87 MG cell line depicting the effect of compounds leading to different pathways of apoptosis [57-60], but none of these revealed the direct effect on cyto-toxicity of such cells in terms of their morphology, cell cycle, proliferation, mitotic catastrophe, apoptosis/necrosis differentiation and molecular docking. However, our work reports the strong anti-proliferative activity of NJRE [its IC50 value being 33.73±3.5, 30.59±3.4 and 28.38±2.9 µg/mL for 24, 48 and 72 h, respectively] even at lower doses on glioblastoma and other cancer cell lines, without harming the normal cells vis-à-vis the synthetic compounds that cause major hazards to the other body cells. Work related to its compounds is in progress in our laboratory and to the best of our knowledge this is our first report of anti-proliferative effect of NJRE on glioblastoma as proved by different bioassays not done earlier. Needless to say this method is more beneficial, cost effective and can be safely employed for clinical trials in future to develop a harmless multi-targeted therapy to manage glioblastoma, as this study has highlighted the probable drug


designing from newer compounds, which is the necessity, considering the gravity of this disease.

Current Cancer Drug Targets, 2017, Vol. 17, No. 1 [7] [8]

CONCLUSION Plant based drugs have been proving to be of utmost importance due to their multi-targeted healing approach. Glioblastoma, a malignant, hyper-vascular and the most lethal kind of brain tumor, and is difficult to manage. Strong anti-proliferative effect of N. jatamansi rhizome extract on U87 MG and other cell lines has been reported for the first time. It caused extensive DNA damage and apoptosis in the U87 MG cell line at doses as low as 30 µg/mL, the response was dose specific and time dependent, this is further validated by the decrease in the expression of caspase 3, caspase 9 and PARP in the treated cells versus control. Moreover, it has least cyto-toxic effect on the normal cell line HEK. The marker compounds of NJRE exhibited drug like properties and docking interactions with various cellular proteins such as: VEGFR2, CDK2, BCL2 and EGFR. The extract therefore, can be used safely in the development of harmless multi-targeted therapy to manage and treat glioblastoma.

[9]

[10]

[11]

[12] [13] [14]

[15]

FINANCIAL SUPPORT Research and Development grant, University of Delhi. SUPPLEMENTARY MATERIAL

[16] [17]

Supplementary material is available on the publishers web site along with the published article. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

[18]

[19]

ACKNOWLEDGEMENTS [20]

Authors are grateful to the University of Delhi, India for providing Research and development grant. HK is indebted to University Grants Commission, India for the award of JRF& SRF.

[21]

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