Characterization and Inception of a Triterpenoid Astrakurkurol, as a

Article Cite This: J. Agric. Food Chem. 2019, 67, 7660−7673

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Characterization and Inception of a Triterpenoid Astrakurkurol, as a Cytotoxic Molecule on Human Hepatocellular Carcinoma Cells, Hep3B Sudeshna Nandi,† Swarnendu Chandra,† Rimpa Sikder,† Saurav Bhattacharya,‡ Manisha Ahir,‡ Debanjana Biswal,§ Arghya Adhikary,‡ Nikhil Ranjan Pramanik,∥ Tapan Kumar Lai,⊥ Michael G. B. Drew,# and Krishnendu Acharya*,† Downloaded via GUILFORD COLG on July 18, 2019 at 12:33:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, WB 700019, India ‡ Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, JD-2, Sector III, Salt Lake, Kolkata, WB 700098, India § Department of Chemistry, University College of Science, 92, Acharya Prafulla Chandra Road, Kolkata, WB 700009, India ∥ Department of Chemistry, Bidhannagar College, EB-2, Salt lake, Kolkata 700064, India ⊥ Department of Chemistry, Vidyasagar Evening College, 39, Sankar Ghosh Lane, Kolkata 700006, India # Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom ABSTRACT: Mushrooms are customary influential sources of pharmaceutically active metabolites. Usually lanostane-type triterpenoids from mushrooms had prospective for cancer disease treatments. Recently, a triterpenoid, astrakurkurol obtained from the fresh basidiocarps of the edible mushroom Astraeus hygrometricus, drew attention as a new cytotoxic therapeutic. The structural stability of this triterpenoid had been established with the amalgamation of density functional theory (DFT) calculations and study of single-crystal X-ray diffraction. To successfully manifest astrakurkurol as a potent cytotoxic therapeutics, a wide apprehension on the molecular and cellular mechanisms underlying their action is prerequisite. On this account, our study was directed to scrutinize the influence of this triterpenoid on human hepatocellular cancer cell model Hep3B. Encapsulating all experimental facts revealed that astrakurkurol had significantly decreased cell viability in a concentration-dependent manner. This effect was unveiled to be apoptosis, documented by DNA fragmentation, chromatin condensation, nuclear shrinkage, membrane blebing, and imbalance of cell cycle distribution. Astrakurkurol persuaded the expression of death receptor associated proteins (Fas), which triggered caspase-8 activation following tBid cleavage. Moreover, tBid mediated ROS generation, which triggered mitochondrial dysfunction and activated the mitochondrial apoptotic events. Astrakurkurol cytotoxicity was based on caspase-8-mediated intrinsic apoptotic pathway and was associated with inhibition at Akt and NF-κB pathway. Astrakurkurol had also inhibited the migration of Hep3B cells, indicating its antimigratory potential. These findings led us to introduce astrakurkurol as a feasible and natural source for a safer cytotoxic drug against hepatocellular carcinoma. KEYWORDS: astrakurkurol, triterpenoid, edible mushroom, liver cancer, apoptosis, migration

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as a whole.4 Hence, considerable efforts were being given to identify therapeutic approaches appreciably from natural sources as they are more active as well as selective, less toxic with limited side effects, and capable of inhibiting growth and invasion of cancerous cells by simultaneously inducing apoptosis in early stage tumors. Mushrooms have been consumed and valued for their inimitable taste, nutraceutical properties, and pharmacon utility. Various medicinal mushrooms known from ancient folklore had reports of use in conventional remedy, and fungal metabolites were popularly used to treat a broad range of diseases.5 The latest studies revealed that there had been an

INTRODUCTION Cancer as a disease has become a huge concern worldwide, usually emerging from aberrant cells with unconstrained division and incursion through blood and lymph systems to other tissues. In the 21st century, overpowering cancer was one of the utmost challenges faced by mankind.1 Among all cancers, hepatocellular carcinoma (HCC) is the biggest recurring malignancy on the globe, which accounts for almost 1 million deaths annually worldwide, and the numbers appear to be increasing substantially in America as well as in other developed western countries.2 Despite major revolutions in modern medicine, the successful diagnosis and effective treatment of cancer still remains a significant challenge.1 Modern cancer treatment involved chemotherapy, surgery, hormone therapy, radiation therapy, and immune therapy based on the stage of cancer progression,3 and most of them pose significant toxic effects to unaffected tissue and the body © 2019 American Chemical Society

Received: Revised: Accepted: Published: 7660

February 20, 2019 June 19, 2019 June 20, 2019 June 20, 2019 DOI: 10.1021/acs.jafc.9b01203 J. Agric. Food Chem. 2019, 67, 7660−7673

Article

Journal of Agricultural and Food Chemistry

MMP-2, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA). Primary antibody against cleaved PARP was shopped from Bioss (Woburn, MA). Apoptosis analysis kit was taken from Biolegend (San Diego, CA). Fetal bovine serum (FBS) and fluorescein-5-isothiocyanate (FITC) were obtained from Invitrogen (Carlsbad, CA). N,N′-Methylene bis acrylamide, TEMED, APS, and Triton X-100 were obtained from Merck, Germany. DCFDA (2′,7′dichlorodihydrofluorescein diacetate) and DiOC6 (3,3′-dihexyloxacarbocyanine iodide) were ordered from Himedia (Mumbai, India). Dimethyl sulfoxide (DMSO) and doxorubicin hydrochloride were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Previously, in our laboratory the compound (astrakurkurol) has been isolated from the basidiocarp of Astraeus hygrometricus, and consecutive crystallization from CHCl3/MeOH and hexane/AcOEt mixtures produced astrakurkurol (drug) in analytically pure forms.13 The purified compound was then dissolved in sterile anhydrous dimethyl sulfoxide (DMSO) to obtain a stock concentration of 20 mM for carrying out the experiments. Crystallographic Measurements for Astrakurkurol. The crystallographic data for astrakurkurol were fetched at 110 K with Mo Kα (λ = 0.71073 Å) radiation on a BRUKER X-ray (three-circle) diffractometer. Data were analyzed with the use of the program SHELXS-97.23 For structural presentation, the MERCURY24 and DIAMOND25 programs have been used. Computational Details. Single point calculations and energy minimizations were achieved using the Gaussian 03 program26 with the B3LYP density functional together with the 6-31G basis set. Starting models were extracted from the crystal structures, but hydrogen atoms have been given only theoretical positions. Cell Culture and Isolation of Peripheral Blood Mononuclear Cells (PBMC). Hep3B (human liver cancer cell line) and nonmalignant human cell line Chang were purchased from the National Centre for Cell Sciences (Pune, India), a national depository of authenticated cell lines. Cells were grown in complete Dulbecco Modified Eagle Medium (DMEM) supplemented with 12% fetal bovine serum (Invitrogen, CA), penicillin (100 μg/mL), and streptomycin (100 μg/mL) in 37 °C humidified atmosphere containing 5% CO2. At a confluency of 80−90%, cells were harvested with trypsin-EDTA (Gibco, NY) and plated for successive experiments. Human whole blood was collected from adult healthy volunteers with prior consent in heparinized BD vacutainer blood collection vials. Lately, 100 mL of whole human blood was diluted in 150 mL of RPMI-1640 medium followed by layering in centrifuge tubes onto 120 mL of Ficoll Histopaque-1077 gradient. After the tubes were centrifuged for 30 min, the whitish buffy coat (peripheral blood mononuclear cells) layer was aspirated and washed twice with PBS. Peripheral blood mononuclear cells (PBMC) were then resuspended in RPMI-1640 containing 10% FBS in 37 °C humidified atmosphere with 5% CO2. Cytotoxicity and Assessment of IC50 Value. To determine the cell growth inhibitory activity of astrakurkurol on Hep3B, PBMC, and Chang cells, WST-1 cell proliferation assay was performed as per the manufacturer’s protocol (Takara Bio Inc., Japan). Briefly, cells were grown in 96 wells of microplate in triplicate at a density of 3 × 105 cells/well to obtain a confluency of about 70%. Prior to adding to the culture medium, astrakurkurol was solubilized in dimethyl sulfoxide (DMSO) followed by serial dilution for various points scaled from 2.1 to 103 μM. The ultimate concentration of DMSO in the equivalent wells did not surpass 1% (v/v) and thereby exerted no effects on cell viability. Treated and untreated cells were incubated with WST-1 reagent for 3 h in a humidified atmosphere. Simultaneously doxorubicin, a familiar anticancer drug maintained as positive control, was also subjected to various concentrations (0.01−1 μM). After 4 h of incubation, the absorbance of the samples was measured with a microplate reader at 450 nm wavelength using a Multiskan GO Microplate spectrophotometer. The percentage of cell growth inhibition was resolved by comparing the cell viability of treated cells with the untreated samples (control cells were considered 100% viable). A dose−response bar graph was formulated to acquire the

upsurge in mushroom use due to the presence of various bioactive substances, which possess antibacterial, antiparasitic, antifungal, antioxidative, antiviral, anti-inflammatory, antiproliferative, antitumor, anticancer, cytotoxic properties, and many more.6−8 In progressing countries like India, inclusive of profuse biodiversity, mushrooms are a privilege for advancement in the discipline of medicine and food.9 The presence of natural bioactive compounds and many secondary metabolites from mushrooms could help in the development of new drugs by reason for their lower cost, low toxicant, and versatile biological resposnse.10 Various clinical trials have been accomplished to evaluate the effects of mushrooms in cancer therapy. Some of the results had already demonstrated a positive outcome.11 Therefore, mushrooms could be regarded as a curative medium in complementary medicine for treating cancer and improving the survival of hepatocellular cancer patients. Astreaus hygrometricus is a delicacy of tribal people found to grow wildly in the lateritic forests of India in association with Shorea robusta.12 It is also recognized as a false earthstar, found helpful for ameliorating burns and lesions, and put to use as a hemostatic agent in Chinese folk medicine. Various heteroglycan sorts of polysaccharides and a splenocyte triggering glucan have been obtained from A. hygrometricus, but few were reported with biological activities.13 From analogous species like Astraeus pteridis and Astraeus odoratus, few lanostane-type triterpenoids with antituberculosis activity had been described.14,15 Extracts from A. hygrometricus with potential antioxidative,12 cardioprotective,16 antidiabetic,17 anti-inflammatory,18 and apoptogenic activities19 were previously reported from our laboratory. Earlier, Lai et al. had reported a new triterpene, astrakurkurol from Astraeus hygrometricus, which exerts promising anticandidal activity.13 Usually drugs derived from biologically active natural products have an enormous contribution to human health,20 and bioactive compounds with helical structures had received special attention as previously Huang et al. (2012) enquired for the role of helicity of anticancer peptides on their bioactivity toward cancer cells.21 In this piece of work, the helical structure of astrakurkurol, stabilized by hydrogen bonds, have been reported as these hydrogen bonds are broadly witnessed as stabilizing interactions for numerous biomolecules and play a pivotal role in drug design. So learning these weak interactions is obligatory for analytical designing of a drug.22 Moreover, studying the helical structure of astrakurkurol will further ignite interest into the role of helicity in cytotoxic activity. The present study essentially focuses on the crystal arrangement of astrakurkurol with various non-covalent interactions and DFT calculations and to unveil the prospective contribution of astrakurkurol in cancer therapy against hepatocellular carcinoma with keen interest to ascertain the possible mechanism that underlies the relationship between astrakurkurol and tumorgenesis.

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MATERIALS AND METHODS

Reagent and Antibodies. Acridine orange (AO), ethidium bromide (EtBr), 4,6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI) were obtained from Himedia (Mumbai, India). Amphotericin B, penicillin, streptomycin, and gentamycin were procured from Himedia (Mumbai, India). WST-1 reagent was purchased from Takara Chemical Co., Ltd., Japan. Antibodies against Bcl-2, Bax, Caspase 8, Caspase 9, Caspase-3, Fas, pAKT (Ser473), 7661

DOI: 10.1021/acs.jafc.9b01203 J. Agric. Food Chem. 2019, 67, 7660−7673

Article


culture plate at a density of 4 × 105. After the cells were treated with the indicated dose (10.3 and 20.6 μM) of astrakurkurol post 24 h incubation, adhered cells were fastened with 3.7% paraformaldehyde for 20 min, and permeabilization was done with 0.1% Triton X-100 at room temperature for 10−15 min. Followed by blocking with 5% BSA for 1 h, cover cells were incubated with monoclonal antibodies consisting of anticytochrome c and anti-p65 (SantaCruz, CA) overnight at 4 °C. Afterward, cells were washed thrice with saline buffer and incubated with FITC (fluorescein isothiocynate)-tagged anti rabbit secondary antibody for 2 h. After being incubated with antibodies, cells in coverslips were rinsed with PBS, and DAPI as a counter stain was added in the slides to examine the cell nuclear morphology. Coverslips were mounted on a clean glass slide by DPX, and images were taken under a fluorescence microscope (EVOS FL, Invitrogen). Cell Death Induction with Caspase Inhibitors. For the caspase inhibition assay, the inhibitors were dissolved in appropriate solvents (DMSO as per manufacturer’s guidelines). Overnight grown Hep3B cells were left to incubate for 2 h with various combinations of inhibitors (25 μM of Z-VAD-FMK and 20 μM of Z-IETD-FMK) before treating with astrakurkurol and incubating for an additional 24 h at 37 °C. Cell viability was observed by WST-1 assay as described in the previous section. Immunoblotting. Preparation of Whole Cell Lysate. For whole cell lysate extraction, astrakurkurol (10.3 and 20.6 μM) treated cells were trypsinized and lysed using RIPA (radioimmunoprecipitation assay buffer) buffer being complemented with protease inhibitor.27 The protein concentration of the samples was quantified using the BCA (bicinchoninic acid) protein assay kit (Merck, Germany). Western Blot Analysis. Cell lysate proteins combined with 1 μL of β-marcaptoethanol and 5 μL of loading dye were heated at 95 °C for 4−5 min. 46 μg of protein from each sample were separated using 10−15% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane (Millipore). Nonspecific binding of the proteins was blocked with 5% skimmed milk for 1 h, and membranes were then incubated with appropriate dilution (1:1000) of specific primary antibodies like GAPDH, Bax, Bcl-2, PARP, Caspase-3, 9, 8, Fas, tBid, Whole Akt, Phospho-Akt (Ser473), and MMP-2 overnight at 4 °C. Then membranes were rinsed thrice with 0.1% TBST, and the blots were reacted with secondary antibodies conjugated to ALP for 2 h. On NBT/BCIP (1:1) addition, the blots were developed and bands were detected. Band density was quantified with the ImageJ program and normalized relative to the housekeeping gene. Validation by Quantitative Real-Time PCR. Total RNA was harvested from Hep3B cells with Trizol reagent (Invitrogen Corp, CA) and purified. Reverse transcription was carried out with 5−10 μg of total RNA using reverse transcriptase and GO Mastermix (MP Biomedicals, U.S.) following the manufacturer’s guideline. To validate the expression results of cDNA obtained from RNA, sequence qRTPCR was carried out using FastStart Universal SYBR Green Master (Roche) in Light Cycler 96 (Roche). The primer sequences used for Bax were sense-5′-TTTGCTTCAGGGTTTCATCC-3′; antisense-5′GTGTTTGGCTTCCTCCAAAG-3′; and for Bcl-2 were sense-5′GAGGATTGTGGCCTTCTTTG-3′; antisense-5′-ACAGTTCCACAAAGGCATCC-3′. All samples were assayed in triplicate, and mean values of these measurements were used to calculate mRNA transcriptions. Fluorescence intensity was measured in real time, and gene (Bax and Bcl-2) expression was defined from the threshold cycle (Cq), and additionally relative expression levels of these two genes were measured and normalized following the protocol described by Bhattacharya et al.28 Bidirectional Wound Healing Assay. Scratch assay is a straightforward and experienced approach to study migration of cancer cells in vitro. 80−90% confluent Hep3B cells were grown in a 12-well plate after which a scratch in a straight line was made with the help of a sterile microtip to form a bidirectional wound. Debris and dead cells were rinsed with PBS, and fresh medium was added followed by treatment with varius concentrations of astrakurkurol. Healing of the wound was quantitated and observed under an

IC50 values with the help of software GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). Clonogenic Cell Survival Assay. The outcome of astrakurkurol on clonogenic survival of cancer cells was determined with a colony formation assay. In brief, cells (1 × 104 cells/well) were seeded in a 12-well plate followed by drug treatment at various (10.3, 20.6, 41.2, and 61.8 μM) concentrations. After overnight incubation, cells were rinsed with saline buffer and supplied with fresh medium and cultured for 3 days in normal conditions. After 3 days, cells were fixed with 3.7% formaldehyde and stained for 15 min with freshly made 0.1% crystal violet. After washing with PBS, colonies formed in each well were photographed. Fluorescence Microscopy and Assessment of Cellular and Nuclear Morphology. Cells (2 × 104 /well) cultured in six-well plates in DMEM for 24 h were treated with astrakurkurol. Treated cells with different (10.3, 20.6, and 41.2 μM) concentrations were stained with a mixture of acridine orange (AO, 5 mg/mL) and ethidium bromide (EB, 3 mg/mL) to distinguish live, apoptotic, and necrotic cells and visualized under a fluorescence microscope (Floid Imaging Station, Life Technologies, Waltham, MA). To examine changes in nuclear morphology, treated (10.3, 20.6, and 41.2 μM of astrakurkurol) and untreated cells were fixed with 3.7% paraformaldehyde and stained with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. The cells were then examined under a fluorescence microscope for nuclear deformities. In both of the microscopic assays, apoptotic morphology was studied and compared to the positive control. Determination of Apoptosis by Annexin V and PI Staining. Quantitative analysis of apoptosis was examined by FITC labeled annexin V and PI kit (Sigma-Aldrich, St. Louis, MO) as per the manufacturer’s protocol. For staining, along with astrakurkurol (10.3 and 20.6 μM), cells (3 × 106/well) were treated with positive control doxorubicin (0.5 μM) for 24 h to observe the comparative effect of our compound with doxorubicin. Post treatment cells were trypsinized and then resuspended in a combined mixture of 300 μL of binding buffer and FITC-conjugated annexin V for 30 min following incubation with propidium iodide for 1−2 min. Stained cells were analyzed using the flow cytometer (BD Biosciences, San Diego, CA) to determine the percentage of early apoptotic, late apoptotic, and necrotic cells. Flow Cytometry Scanning for Cell Cycle Phase Distribution. The 5 × 106 Hep3B cells were plated in a six-well culture plate for 24 h and treated with two different dilutions (10.3 and 20.6 μM) of astrakurkurol. Post treatment trypsinized cells were fixed in chilled 70% ethanol and incubated for 24 h at 4 °C. Fixed cells were incubated for 10 min in dark and stained with freshly prepared PI solution (50 μg/mL propidium iodide and 10 μg/mL RNAase). Cell cycle phase distribution of nuclear DNA was extracted using a flow cytometer (BD Biosciences, San Diego, CA), and Flowjo software is used for data analysis. Measurement of Intracellular ROS Production. The intracellular reactive oxygen species (ROS) levels were quantified using fluorescent dye DCFH-DA (2′,7′-dichlorofluorescein diacetate). To determine drug-induced intracellular ROS production, Hep3B cells were cultured in a six-well plate with and without varied drug concentrations (10.3 and 20.6 μM). After treatment, cells were harvested and stained with ROS indicator, 1−2 μM DCFDA, in the dark at 37 °C for 15 min. The fluorescent intensity produced in cells was measured by BD FACS Verse. Detection of Changes in Mitochondrial Membrane Potential. Fluorescent cationic dye DiOC6 was used to measure the disruption of mitochondrial membrane potential. After treatment with astrakurkurol, Hep3B cells were trypsinized, rinsed with PBS, followed by 1−2 min incubation with 0.2 μM DiOC6 at 37 °C. Changes in mitochondrial distribution pattern were analyzed with a flow cytometer (BD Bioscience, San Diego, CA), and the mean fluorescence intensity of treated cells was compared to an untreated set of cells using BD CellQuest Pro software. Fluorescence Imaging. For fluorescence imaging, Hep3B cells were cultured in 22 mm square glass coverslips in a six-well tissue 7662


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Figure 1. Crystal structure elucidation and frontier orbitals of astrakurkurol. (A) Schematic molecular structure of the compound. (B) 1D supramolecular helical chain formation of astrakurkurol along the c-axis. (C) 3D packing of astrakurkurol along the c-axis. Plots of (D) HOMO; (E) HOMO−1; and (F) LUMO. inverted microscope (Floid Cell Imaging Station, Life Technologies, U.S.) after 24 h of treatment. The time required to fill the wound edges was calculated by comparing the images from time 0 to 24 h. The percentage of cell migration of control was taken as 100%, and wound closure for treated cells was correlated with respect to control set. Transwell Migration Assay. To evaluate cell invasion ability, transwell migration assay was carried out. 8 mm pores of cell culture inserts (BD Biosciences, Sparks, MD) were settled in a 12-well companion plate. For the upper half of the insert, which was placed inside the chamber, 2 × 105 cells were resuspended in 500 μL of serum-free medium, whereas the lower chamber was filled with DMEM supplemented with 10% FBS as chemoattractant. The plate was then incubated in culture conditions. After 24 h, inserts with cells were washed with PBS, fixed in 3.7% formaldehyde, permeabilized with methanol, and nonmigratory cells remaining on the upper half of the chamber were removed by scrubbing with a cotton tip applicator. The migrated cells on the lower surface of the inset were stained with 0.1% Giemsa for 30 min followed by PBS wash and visualized under a microscope. The statistics of migrating cells were obtained by imaging and counting five randomly selected fields per well, and the percentage of cell migration was expressed as an average number of cells per microscopic field.

of refinement and both intra- and intermolecular hydrogenbonding geometries are noted in Tables 1 and 2. Table 1. Crystal Data and Details of Refinement Obtained from the Single-Crystal X-ray Diffraction Study of Astrakurkurol Astrakurkurol chemical formula formula weight (M) crystal system space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) Z2 crystal size temp (K) DC (g cm−3) μ(Mo Kα) (mm−1) F(000) GOF on F2 R1, wR2 [I > 2σ(I)]a

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STATISTICAL ANALYSES All statistical analyses were implemented using the software GraphPad Prism version 5 (San Diego, CA). Data are presented as mean ± standard deviation (SD) unless mentioned otherwise. One-way and/or two-way analysis of variance was performed to analyze the data followed by Dunnett’s (to compare between control groups versus all of the treated groups) test and Bonferroni’s post hoc (to compare between selected groups) among multiple comparison tests. A P-value < 0.001 was set to denote a statistically significant difference unless mentioned otherwise.

a

C32H53O3 485.74 monoclinic P2(1) 10.978(3) 7.6619(19) 17.468(4) 90.00 102.955(3) 90.00 1431.9(6) 0.70 × 0.70 × 0.20 110(2) 1.127 0.070 538 1.026 R1 = 0.0383 wR2 = 0.1005

R = ∑||F0| − |Fc||/∑|F0|; wR(F2) = [∑w(|F0|2 − |Fc|2)2/∑w|F0|4]1/2.

Astrakurkurol crystallized in the monoclinic space group P2(1) as colorless needles. The single-crystal X-ray structure revealed that hydrogen bonds hold a vital role in stabilizing the compound structure. The compound exhibits a remarkable supramolecular assembly in the solid state. As depicted in Figure 1B, the astrakurkurol molecules were organized in a unique way to construct a 1D supramolecular right helical chain along the c-axis. The C(7)−H(7A)···O(3) hydrogen

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RESULTS Crystal Structure Description of Astrakurkurol. Figure 1A depicts the molecular structure of astrakurkurol with atom numbering scheme. Crystallographic data together with details 7663


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Journal of Agricultural and Food Chemistry Table 2. Geometry of Strong Hydrogen-Bonding Interactions in the Crystal Structure of Astrakurkurol D−H

A

H···A (Å)

D···A (Å)

D−H···A (deg)

symmetry code

O(3)−H(3) C(7)−H(7A) C(11)−H(11A) C(3)−H(3A) C(8)−H(8C) C(23)−H(23A) C(6)−H(6A)

O(2) O(3) O(2) O(1) O(1) O(3) O(2)

2.254 3.09 2.73 2.57 2.60 2.67 2.64

3.078(2) 3.774(2) 3.035(2) 2.851(2) 2.877(2) 3.038(2) 2.931(2)

167.0 127.8 98.3 96.4 96.9 101.6 97.4

1 − x, 1/2 + y, −z 1 − x, 1/2 + y, 1 − z x, y, z x, y, z x, y, z x, y, z x, y, z

and Chang cells (Figure 2A and B). To further establish our finding, clonogenic assay was performed. The clonogenic cell survival method usually regulated the capability of a cell to retain its reproductive viability by forming a clone or a large colony.31 Our findings showed that untreated cells proliferated indefinitely, whereas at a dose of 20.6 μM, almost one-half of the colonies were decreased and further reducing distinctly at doses of 41.2 and 61.8 μM of astrakurkurol (Figure 2D). Thus, astrakurkurol strongly affected the proliferation of invasive cells in vitro. Astrakurkurol Induces Apoptotic Changes and Cell Cycle Arrest in Hep3B Cells. To resolve if the observed decrease in cell viability was induced due to apoptosis, morphological studies were carried out to investigate the mode of cell death caused by astrakurkurol using fluorescence microscope. Initially exposing the cells to several dosages of astrakurkurol for 24 h, we analyzed the living cells and cells dying of necrosis or apoptosis through AO/EtBr dual staining. Consistently green fluorescence living cells with normal morphology were observed in the control group, and in the experimental group more cells were EtBr positive and showed yellow green fluorescence, indicating early apoptotic phase, and in higher dose the amount of apoptotic cells increased with orange margination around the cells with nuclear fragmentation, shrinkage, apoptotic body formation, chromatin condensation, and membrane blebbing (Figure 2E). Consistent with the above results, astrakurkurol treated cells were then exposed to DAPI for nuclei staining. With increasing concentration of astrakurkurol, the nuclei underwent shrinkage, membrane blebbing, condensation, and fragmentation, which are morphological changes regarded as typical characteristics of early apoptosis (Figure 2F). Both of these staining procedures were carried out in the presence of a positive control doxorubicin (Figure 2E and F). After screening various dosages with a preliminary study, we chose two doses below the IC50 values, i.e., 10.3 and 20.6 μM of astrakurkurol to carry out further investigation. To further reinstate the findings obtained from microscopy, apoptotic cell death was validated by Annexin V-FITC and PI double staining flow cytometry. It was evaluated that the early apoptotic population in control cells was 1.42%, which escalated to 42.11% and 53.81% when treated with 10.3 and 20.6 μM astrakurkurol, respectively, whereas the statistics of late apoptotic population was found to be 1.12% in control cells and 6.83% and 4.76%, respectively, in treated sets. As a positive control, Hep3B cells were treated with a standard chemotherapy drug, doxorubicin, which showed 63.08% of the total apoptotic population (Figure 2G). The PI positive cells, which represented the necrotic population, were not observed to increase evidently on exposure to astrakurkurol. The results from AnnexinV-FITC/PI double staining clearly suggested the possible apoptotic activities of astrakurkurol. The mechanism

bonding leads to the formation of the helical architecture. Figure 1C shows the 3D packing of the adjacent helical chains along the c-axis. Theoretical Calculations. DFT Optimized Structures and Relative Stabilities. To figure out the delicate interplay of cooperative non-covalent interactions, which stabilize the structure of astrakurkurol in the solid state, we have completely amended the structure. The essential feature of the packing involves the formation of hydrogen bonds involving the enolic −OH group; the molecules are linked via a screw axis with a strong hydrogen bond between O(3)−H(3) and O(2) (1 − x, 1/2 + y, 1 − z) of 3.078(2) Å. The molecule also shows weak C−H···O interactions, which are listed in Table 2. Single point calculations were executed to evaluate the effect of the O−H···O hydrogen-bond formations in the molecule. A calculation was carried out for two molecules attached via one hydrogen bond and for one molecule separately. The energy difference E [(two molecules) − 2*(one molecule)] was −12.12 kcal mol−1. Of course this energy difference is not due entirely to the O−H···O hydrogen bond as the interactions between other atoms in the two molecules will have some effect. The molecule was then energy-minimized, and the geometry showed little difference from the starting model. Molecular Orbitals. The frontier orbitals of the compound were then investigated. The HOMO, HOMO−1, and LUMO were shown in Figure 1D−F. As might be expected in this structure, the HOMO is concentrated on the double bond, as indeed is the LUMO. By contrast, the HOMO−1 is concentrated on the oxygen atoms at the other end of the molecule. The other frontier orbitals from HOMO−4 to LUMO+4 are spread out over many atoms. Growth Inhibitory Effect of Astrakurkurol on Hep3B Cells. To inquire about the therapeutic impact of astrakurkurol, Hep3B cells were plated, which display high levels of proliferation and migration. Initially the cytotoxicity of astrakurkurol on Hep3B cells was evaluated by the WST-1 assay kit. As shown in Figure 2A, the survival curve exhibited a remarkable inhibition on the survival of Hep3B cells in a dosedependent manner. Within 24 h, after exposure to various dosages (ranging from 2.06 to 103 μM) of astrakurkurol, cell viability was reduced, whereas at the highest dose, that is, 103 μM, the drug shows 100% cell death. The IC50 values, that is, the concentration at which 50% cell growth has been inhibited at 24 h for human hepatoma cancer cell, were found to be 22.6 μM. Alongside the cells were also incubated with an accepted anticancer drug doxorubicin (as positive control) for 24 h, which showed an IC50 value to be approximately 0.3 μM (Figure 2C), which is comparable with the results published previously.29,30 Contrarily, the percentage of cell viability of Chang and PBMC was convincingly higher when treated with astrakurkurol. These results reflect that Hep3B cells were majorly susceptible to astrakurkurol as compared to PBMC 7664


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Figure 2. Astrakurkurol inhibits cell viability and induced apoptosis in Hep3B cells. (A) The cytotoxic effect of astrakurkurol was measured by WST assay against Hep3B and Chang cells for 24 h at the indicated concentration. The cell viability of control set was considered 100%. (B) Analysis of percentage of cell survival by WST assay on PBMC (peripheral blood mononuclear cells) at various doses of astrakurkurol for 24 h. (C) Graphical delineation of the percentage of cell death of Hep3B cells by WST assay at indicated dose of doxorubicin (positive control). (D) The colony-formation assay demonstrated a dose-dependent decrease in Hep3B cell proliferation when exposed to astrakurkurol. Results represented four concentrations of astrakurkurol treatments for 3 days. (E) Acridine orange/ethidium bromide staining was carried out to reveal and discriminate viable and apoptotic cell morphology of astrakurkurol treated Hep3B cells for 24 h. Cells were also treated with doxorubixin (DOX) as positive control of apoptosis induction. Green live cells show normal morphology in control; yellowish green early apoptotic cells show nuclear margination and chromatin condensation; and slightly orange margination of cells indicates later apoptotic cells with more morphological damage. (F) Nuclear morphological changes on astrakurkurol treated Hep3B cells were captured by staining with DAPI. Hep3B cells were treated with indicated concentrations of astrakurkurol and doxorubicin for 24 h. (G) Apoptotic cells were measured by Annexin V/PI staining with a flow cytometer. Cells treated with indicated doses of astrakurkurol and doxorubicin for 24 h. Criteria were set to distinguish between early apoptotic (lower right quadrant), late apoptotic (upper right quadrant), and necrotic (upper left quadrant) cells at two doses of astrakurkurol. Panel at right shows the bar diagram displaying percentages of early and late apoptotic cells. (H) Cell cycle distribution was analyzed by PI staining in Hep3B cells with and without astrakurkurol treatment for 24 h using a flow cytometer (left panel). Histogram showing the distribution of cells in Sub-G1, G0/G1, S, and G2/M phases. The data are presented as mean ± SD of three replicates of independent experiments.

underlying the observed astrakurkurol-mediated apoptosis was further scrutinized and reaffirmed in the Hep3B cell line. To enquire whether the antiproliferative effect of astrakurkurol on Hep3B cells is partially associated with cell cycle arrest, treated cell were stained with PI, and changes in different phases of cell cycle were analyzed with the help of FACS. As illustrated in Figure 2H, astrakurkurol increased the area of the peak corresponding to hypodiploid (apoptotic sub

G0) DNA content in Hep3B cells. Treating cells with increasing concentrations of astrakurkurol increased the number of cell population in the G0/G1 phase. This increase in the number of cells in the G0/G1 phase (51.5% in control, 59.5% at 10.3 μM, and 63.2% at 20.6 μM) was escorted with a concordant decrease in the amount of cell population in G2/M phase (29.66% at control set, 19.5% at 10.3 μM, and 14.79% at 20.6 μM) with almost no change in the S phase (17.68% in 7665


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Figure 3. Astrakurkurol triggers extrinsic apoptotic signaling molecule and caused tBid-mediated activation of proapoptotic molecule Bax. (A) Dose-dependent impact of astrakurkurol on protein expression of extrinsic apoptotic signaling molecule including Fas and cleaved caspase-8 was evaluated by Western blot in treated Hep3B cells (right panel). Change in expression of band intensities was quantified using the ImageJ program after normalizing with GAPDH that served as internal loading control (right panel). (B) Hep3B cells were pretreated for 2 h with indicated inhibitors Z-VAD FMK and Z-IETD-FMK before treatment with atrakurkurol. The cell viability was determined using WST-1 reagent. (C) Western blot analysis of tBid, Bcl-2, and Bax protein levels in astrakurkurol treated and untreated cells. (D) Bar graph obtained from qRT-PCR analysis depicts the changes in the RNA expression of Bax and Bcl-2. The results are expressed as the mean ± SD of three independent experiments. The asterisk indicates a notable difference from the control group (*P < 0.01, **P < 0.001, and ***P < 0.0001 versus control).

control and 17% in 10.3 and 20.6 μM). From this result, it has been concluded that astrakurkurol induces a G0/G1 phase arrest, which corresponds with the inhibitory effects of astrakurkurol against Hep3B cells. Astrakurkurol Prompts the Expression of Extrinsic Signaling Molecules To Trigger Apoptosis in Hep3B Cells. In hepatocellular carcinoma cells, the loss of sensitivity (a hallmark of cancer) occurs through several processes, which include resistance to apoptosis via death receptors (DRs).32 Literature study revealed that extrinsic pathway is conciliated by death receptors and corresponding proapototic ligands, which then activate the death receptors followed by the activation of downstream signaling molecules including FADDR and caspase-8 that escorts apoptosis induction.33 Thus, in our investigation, we have tried to dig into the mode of action of astrakurkurol-mediated cell apoptosis. To endorse the regulation of death receptor-mediated apoptotic proteins, expression levels for the proteins Fas and caspase-8 were determined (Figure 3A), which showed an increased amount of proteins in both of the treated sets in comparison to control Hep3B cells. Further as a preparatory molecular approach to investigate the connection of caspase-8 in the apoptotic cascade of Hep3B cells, we analyzed the activity of an irreversible pan-caspase inhibitor, Z-VAD-FMK, and a specific caspase-8 inhibitor, Z-IETD-FMK. We observed that pretreatment with both the inhibitors has procured generous protection from cell death in astrakurkurol treated cells, whereas in the absence of these inhibitors no protective effect was observed (Figure 3B). Strikingly, the specific caspase-8 inhibitor had distinctly caused a higher protective effect of around ∼25%, whereas whole caspase inhibitor had a minimal protective effect of around ∼15% against astrakurkurol treatment. As a more substantial protective effect was found

to cause by Caspase-8, it was suggested that caspase-8 might exert unusual roles in Hep3B cells. Caspase-8 Mediates Cytochrome c Release and Drives Mitochondrial Pro-apoptotic Proteins. Studies revealed that an essential function of activated caspase-8 is to cleave Bid (cytosolic BH3 interacting domain) to truncated Bid (tBid), which then translocated to mitochondria and, through immediate connection with antiapoptotic members of the Bcl-2 family, releases proapototic signal Bax to create pores in the mitochondrial membrane leading to the dispensation of cytochrome c and initiates caspase cascade activation and escalates intrinsic apoptosis pathway.34 Hence, with this perception, we were prompted to explore the role of activated caspase-8 in the presence of astrakurkurol in Hep3B cells. Starting with Western blot, expression level of tBid was detected in higher concentrations of astrakurkurol treatment (Figure 3C), which further instigated us to examine the Bcl-2 family representative that might collaborate with tBid to induce ROS generation and mitochondrial outer membrane permeabilization. We employed real-time PCR as well as Western blot to detect the expression level of both pro- and antiapoptotic members of the Bcl-2 family. The real-time PCR and Western blot data showed an elevated expressivity of Bax (proaptotic) and downregulation of Bcl-2 (antiapoptotic) in a concentration-dependent manner (Figure 3C,D). This apoptotic condition bringing change in the ratio of pro and antiapoptotic Bcl-2 family members has promoted prompt ROS generation as illustrated in Figure 4A with DCFDA dye, which further rapidly dropped the mitochondrial energy of treated sets, as reflected by a decrease in fluorescence as well as shifting of peak in the left side that illustrates that lesser cells retained DiOC6 in their mitochondria (Figure 4B). ROS generation and mitochondrial dysfunction eventually lead to 7666


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Figure 4. Astrakurkurol initiates mitochondrial intrinsic apoptotic pathway and regulates phospho-akt status and intracellular location of NF-κB p65 subunit. (A) Detection of intracellular ROS level at the indicated doses of astrakurkurol measured by a flow cytometer (left panel). Bar graph represented the amount of DCF fluorescence generated in Hep3B cells (right panel). (B) Histogram overlay represents the changes observed in mitochondrial membrane potential (ΔΨm) by staining the astrakurkurol treated cells with DiOC6 (left panel). Bar diagram representing percentage of cells labeled with DiOC6 dye post astrakurkurol treatment (right panel). T1 represents 10.3 μM and T2 represents 20.6 μM. (C) Immunocytochemistry study to point out the increased expression of Cytochrome C1 in the cytosol upon astrakurkurol treatment in Hep3B cells. Control and treated cells were stained with anti-cytochrome c and further stained with FITC-tagged secondary antibody and counter stained with DAPI. (D) The expression level of cleaved caspase-9, 3, and PARP in the control and treated cells at transcriptional level. GAPDH treated as internal loading control (left panel). Expression of band intensity was represented using the ImageJ software (right panel). (E) Expression levels of phospho-Akt and total akt at the indicated doses of astrakurkurol. GAPDH used as loading control (left panel). Graphical representation of band intensities using the ImageJ program and normalized to the respective loading controls (right panel). (F) Immunofluorescence images of astrakurkurol treated Hep3B cells and subsequently stained with anti NF-κB antibody. The anti NF-κB secondary antibody was FITC tagged (green), and nuclei were stained with DAPI.

apoptosis pathway, we checked the expression levels of some vital proteins including Caspase-9, -3, and PARP (Figure 4D). Data obtained from blots represented distinct overexpression of active caspases-9 and -3 that relatively leads to the cleavage of target protein poly(ADP-ribose) polymerase (PARP) in treated cells. All of these outcomes together help to conclude that exhilaration of the mitochondrial arm of the apoptosis pathway is essential for the initiation of astrakurkurol modulated apoptosis followed by a death receptor stimulus. The Caspase-8-conciliated spliting of the pro-apoptotic Bcl-2

the spilling out of cytochrome c. The immunofluorescence data disclosed that there was an observable diffused staining pattern in most treated cells consistent with a translocation of Cyt c into the cytosol, whereas Cyt c showed a dotted pattern in control cells, persisting with its localization within the mitochondria (Figure 4C). Meanwhile, the margination as well as the condensation of chromatin of the nucleus were also noticed by exposing the cells with DAPI. Further, to contemplate the finding of whether caspases were involved in astrakurkurol-induced apoptosis to amplify the intrinsic 7667


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Figure 5. Astrakurkurol suppresses Hep3B cells migration in vitro. (A) Hep3B cell migration inhibited in a wound healing assay on treatment with different concentrations of astrakurkurol. Microscopic images were captured at 0 and 24 h after creating the wound (left panel). Line diagram represents the percentage of migratory cells (right panel). (B) Phase contrast images showing the Giemsa stained Hep3B cells that migrated through the micropore membrane in the control and treatment with different concentrations of asrakurkurol (left panel). Line diagram displaying the percentage of migratory cells after 24 h (right panel). (C) SDS-PAGE followed by Western blot represents the changes in MMP-2 expression levels for indicated concentrations of astrakurkurol (left panel). Changes in expression were defined after normalizing with loading control (right panel). Images shown are representative of three independent experiments. *P < 0.01, **P < 0.001, and ***P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparison test (to compare between control and all of the treated groups).

to control, whereas the level of total akt levels persisted almost the same (Figure 4E). Thus, downregulated Akt could not activate the NF-kB activity, which was further verified by the images obtained from NF-kB-p65 immunostaining in astrakurkurol treated cells along with control cells and counter staining the cells with a nuclear marker, DAPI. The immunocytochemistry results open up that there was a marked decrease in nuclear fluorescence of treated sets as shown with negligible or least fluorescence at 20.6 μM treatment, unlike the untreated cells (Figure 4F). Downregulation of phosphoakt and negated nuclear translocation of NF-kB pointed to the fact that astrakurkurol might induce apoptosis of Hep3B cells via diminishing the cell survival pathways. Astrakurkurol Suppresses in Vitro Migration and Invasion of Hep3B Cells. In the interest of interrogating the

family member Bid is considered as the prime bridging element between the two linking arms of the apoptotic pathway. Astrakurkurol Attenuates Phospho-akt Status and Intracellular Localization of NF-kB p65 Subunit To Incite Apoptosis. To inquire about the underlying molecular mechanism trailing the astrakurkurol-induced caspase-8mediated intrinsic mitochondrial apoptotic pathway, the status of Phospho-akt and localization of NF-kB p65 subunit were investigated. Akt, a vital regulator for survival and proliferation of human cells, is a prime upstream factor that activates and regulates the NF-kB activity via phosphorylation of p65.35 We initially determined the amount of protein expression of phospho-akt in treated as well as untreated cells. The blots reflected that the expression level of phosphorylated akt has been downregulated post treatment with astrakurkurol relative 7668


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helical conformations of pro-apoptotic (Bax) and antiapoptotic (Bcl-2, Bcl-XL) proteins,46,47 so compounds with helicity are quite effective in targeting specifically Bcl-2 or BclXL function in the tumor cells. These helical anticancer compounds binding with anti or pro apoptotic proteins will disrupt the heterodimerization of Bax/Bcl-2 and promote the release of cytochrome c from mitochondria, which ultimately causes apoptosis.41−44 The main finding of our study is the establishment of triterpenoid, astrakurkurol, as a cytotoxic drug which propagates apoptosis of hepatocellular carcinoma, Hep3B, cells by death receptors cross-linking to proapoptotic sequential events in mitochondria. Our findings revealed that astrakurkurol acts directly on Hep3B cells to promote cytotoxicity in a mean that induces apoptosis. The cytotoxicity induced by the drug was tested using the WST-1 assay system, preferred over the conventional MTT assay to negate the artifacts produced due to the poor solubility of the MTT formasan complex in water.48 As indicated in WST and colony formation assays, astrakurkurol emerged as a strong growth inhibitor and is selectively toxic to cancer cells with almost no cytotoxicity toward the PBMC and Chang liver cells. As stated by Blagosklonny (2005), an ideal anticancer agent ought to be efficient in killing cancerous cells and selective toward normal cells.49 Several cytotoxic anticancer agents were delineated to cause cell death of tumor tissues by activating apoptosis.50 Hence, induction of apoptosis was described to be a potential mechanism of chemoprevention and chemotherapy. To further affirm the apoptosis-induced cell death by astrakurkurol, typical apoptotic changes were observed in both cellular and nuclear morphology by different staining methods. It has been recognized that, in addition to plasma membrane blebbing and nucleus and cytoplasmic condensation, apoptotic cells can also be characterized on the basis of loss of plasma membrane asymmetry and attachment.51 Thus, to validate, the microscopic data were justified with an increase in cell population for Annexin V-FITC positive or both Annexin V/PI positive, indicating cells had died by apoptosis. Previous studies explained that initiation of apoptosis and cell cycle arrest are recommended as standard mechanisms for the cytotoxic efficiency of chemotherapeutic drug, which were obtained from herbal medicine. Generally, in cancerous cells, arrest in cell cycle could trigger proliferation inhibition and apoptosis.52 In astrakurkurol-induced apoptosis, an underlying mechanism appeared to be firmly connected with its effect on the cell cycle. Usually chemopreventive agents caused cell cycle arrest at the G0/G1 or G2/M phase, but comparatively less knowledge has been established regarding mechanisms that prevent the progress within the S phase.53 We detected astrakurkurol to induce arrest at the G0/G1 cell cycle phase as similarly Hispolon caused arrest in the cell cycle of human leukemia cells.54 Later, to explore the anticancer competency of astrakurkurol, we are keen to discover the detailed molecular mechanism behind the induction of apoptosis in Hep3B cells. As we know, apoptosis is accomplished through two leading pathways: the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway.55 In the death inducing signaling pathway upon stimulation of the FAS/TNFR-1 pathway, caspase8 gets activated and releases into cytosol to cleave various substrates. Of various substrates, proapoptotic Bcl-2 family member Bid is the prime substrate of caspase-8, which was cleaved into truncated bid (tBid), translocated to

influence of astrakurkurol on cell migration and invasion, in vitro bidirectional wound healing, transwell migration, and Western blots for MMP-2 were conducted. As illustrated in Figure 5A, the wound enclosure of treated cells was indicatively suppressed by astrakurkurol, respectively, at a low dose of 3.43 μM. On the contrary, untreated cells display the fastest capacity to fill the gap at the 24 h time point. These findings therefore highlighted the fact that astrakurkurol suppresses the in vitro mobility of cancer cells at a low dose of 3.43 μM, which is even lower than the effective apoptotic dose of 10.3 μM. To further endorse the effects of astrakurkurol, the transwell in vitro migration assay was conducted. On the basis of the findings obtained from Figure 5B, we observed that a lesser number of Hep3B cells was found to have migrated to the lower half of the transwell insert when treated with the above-mentioned doses, indicating that the rate of migration through the transwell membrane had decreased as the dose increased. Further, the tumor metastasis related marker protein, MMP-2 expression at the translational level, was observed to be downregulated at concentrations of 10.3 and 20.6 μM (Figure 5C). Taken together, all of these data sets highlighted the activity of astrakurkurol in cell migration inhibition.

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DISCUSSION Terpenoids account for a substantial source of compounds, which are convenient in the prevention and therapy of a handful of diseases36 as well as drug discovery,37 and terpenoids extracted from mushroom have been kindred with diverse pharmacological activities, specifically anticancer activity.38 Various preclinical studies have presented comprehensive documents that both natural occurring and synthetically derived triterpenoids together own therapeutic and chemopreventive activity against different cancers.36 Several research groups further added that any triterpene compound with helicity will enumerate their anticancer activity.39 Higuchi and co-workers reported the cytotoxic behavior of helical metallosupramolecular polymers and highlighted the importance of the presence of helicity in a potent anticancer compound for chiral recognition of DNA.40 Our recent work emphasized to look into the X-ray crystal structure of this natural lanostane-type triterpenoid, which reveals appealing cooperative non-covalent interactions. DFT calculations of the compound indicate that in the solid state, non-covalent interactions have a crucial role in stabilizing the structure of astrakurkurol. To identify the compositions of the relevant HOMOs and LUMOs, molecular orbital calculations have been utilized. The hydrogen-bonding interactions further facilitated the formation of a compelling helical architecture in 1D. As DNA has a double helical structure, it easily forms a conjugate with a helical potent anticancer agent, and the conjugate is usually stabilized by favorable electrostatic interactions between the DNA and the anticancer compound. Our compound, astrakurkurol, also exhibits a helical conformation similar to DNA, and eventually this strong binding between the astrakurkurol and DNA might inhibit DNA replication, thereby eventually leading to cell death. Hence, it can be suggested that helicity observed in the structure of astrakurkurol is an important structural requirement to emerge as a potent cytotoxic agent. An alternate mode of action of helical anticancer agents was also provided by several researchers.41−45 Nuclear magnetic resonance imaging and X-ray crystallography have revealed the existence of α7669


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study, we have hypothesized that in the apoptosis process, a practical association could persist amidst astrakurkurol and PI3K/Akt/NF-kB pathways, and inactivation of these pathways may encourage HCC cells apoptosis. This supported us to interrogate the status of phosphorylated-akt, and we observed that expression of phospho-akt was down-regulated while the total akt levels remain the same on incubation with astrakurkurol. Previously, in MA-10 mouse Leydig tumor cells, Cordycepin, obtained from the mushroom Cordyceps militaris, was found to elevate ROS production and induce apoptosis by way of down-regulating the p38 MAPK and PI3K/AKT signaling pathways at moderate concentration levels.65 Similarly, the PI3K/AKT pathway was found to be inhibited by curcumin to incite apoptosis in p53-null hepatoma cells.66 As we have already observed that the band intensity of phospho-akt has been diminished on astrakurkurol exposure, so we were fascinated to check if it possesses any effects on NF-κB. Heterodimer NF-κB includes two subunits, where the transactivating domain has been placed at the C terminal of p65. On stimulating the signaling route of NF-κB, the p65 subunit gets translocated to the nucleus and develops tumor advancement with antiapoptotic processes.67 Our findings revealed astrakurkurol efficiently restricted the nuclear translocation of the p65 subunit of NF-κB, which indeed suggested the efficiency of astrakurkurol in suppressing the progression of cancer cells via inhibiting the Akt signaling route. Astrakurkurol was further assessed for its antimetastatic potential. Cancer metastasis explains the spreading of cancerous cells to other tissues and organs, which leads to the demise of many patients. Metastasis and invasion of solid tumors demand cessation of matrix metalloproteinases (MMPs) and basement membrane with the potentiality to deteriorate the extracellular matrix of the invading surrounding tissues.68 HCC was reported as a malignant tumor whose invasion and metastasis were causes of multigene interactions. Of two matrix metalloproteinases, MMP-2 is proficient in breaking down most of the components of extracellular matrix as well as closely correlated in promoting tumor recurrence and metastasis.69 In vitro, we found that astrakurkurol inhibited cancer cell migration as well as invasion via significantly downregulating the MMP-2 protein as well as by decreasing the migration rate of Hep3B cells toward the center of the wound on increasing the concentration of astrakurkurol. Moreover, a similar pattern has also been observed in the in vitro transwell migration assay where a lower number of migrated cells was accumulated in the lower half of the transwell. These studies discussed so far perspicuously re-elect our findings that astrakurkurol unveils a influential antimigratory property in Hep3B cells along with its notable apoptotic nature. To summarize, our experimental setup illustrates that astrakurkurol-induced cleavage of Bid by caspase 8 and the successive outer mitochondrial membrane association of tBid are two explanatory events that trigger up Bid, enabling the engagement of the mitochondrial pathway during death receptor-mediated apoptosis in Hep3B cells. This pure compound has simultaneously assisted in inducing apoptosis and obstructing migration in Hep3B cells without proclaiming any noteworthy cytotoxicity in normal cells. These findings impart a perception into the insight on molecular mechanisms of astrakurkurol-induced apoptosis in HCC cells and introducing this triterpenoid as an effective inducer of apoptosis and antagonist of metastasis, which would be beneficial for the further utilization of many medicinal fungi

mitochondria causing cytochrome-c spillage from mitochondria, which commences the stimulation of the caspase cascade and augments the intrinsic apoptosis pathway,56 thereby portraying a connecting bridge between two apoptotic pathways. Our present study had evidently demonstrated that Hep3B cells treated with astrakurkurol had stimulated the death receptor Fas with activation of downstream players like caspase-8 and tBid as evident from the immunoblots. Further, at the preliminary level, to assess the canonical involvement of caspase-8 in apoptosis, we were startled to detect that pretreatments with specific caspase-8 inhibitor had consequently promoted lesser cell death in contrast to whole caspase inhibitor. Lately tBid cooperated with Bax and displaced Bcl-2 (antiapoptotic marker), which is evident from the diminished expression of Bcl-2 and marked increase of Bax in both transcriptional and translational level. The overexpression of Bax and displacement of Bcl-2 further caused ROS generation, which is consistent with previous reports observed in Jurkat cells as well as pancreatic β-cells incubated with doxycycline and high glucose medium, respectively.57,58 In this context, the ROS generation was determined using a fluorescent probe, DCFDA, and the peak was observed to be shifted to the right. It is widely familiar that ROS production is required in the opening of the mitochondrial transition pore as well as mitochondrial membrane dysfuction.59 Dysfunction of the mitochondrial membrane causes exemption of cytochrome c from mitochondria into the cytosol and recruits procaspase 9 for apoptosome complex formation and activate executioner caspase 3 to bring about cell death.60 While measuring the mitochondrial membrane integrity, astrakurkurol was found to significantly decrease the fluorescence intensity of DioC6 as well as initiated the spillage of cytochrome c into cytosol, which was clearly evident from the staining pattern of immunolabeled cytochrome c in treated and untreated sets, signifying the commencement of the intrinsic apoptotic pathway. Additionally, the apoptotic control by astrakurkurol had been validated by showing amplified expression levels of caspases-9, -3, and downstream effector cleaved PARP. In accordance with our compound’s mechanism of action, Cordycepin, a bioactive compound isolated from Cordyceps militaris also induced human liver cancer (HepG2) cell apoptosis in caspase-dependent pathways via activating caspases, developing synergy between Fas and FADD, and by harmonizing the protein levels of Bid and tBid.61 Cordycepin with broad antitumor activity has integrated mitochondria involved apoptosis in gastric cancer cell (SGC7901 cells) by controlling mitochondrial pathways and triggering a death receptor like DR3, which further stimulated the PI3K/Akt protein expression as well as depolarized the mitochondrial membrane potential (MMP).62 The mechanistic pathway of astrakurkurol can also be corroborated with Suillin, a prenylphenol obtained from the mushroom Suillus placidus also dissolved in DMSO that has notably prevented cell proliferation and elicited apoptosis in human hepatoma HepG2 cells by way of both death receptor and mitochondrial pathway.63 Various studies explained that enhanced PI3K/Akt activation can accelerate the proliferation as well as negation of HCC cells from apoptosis and thereby turn into an absolute prognostic index for HCC patients. Additionally, the Akt signaling pathway also advances the awakening of NF-kB signaling pathway, which restricts apoptosis by inducing upregulation of principal antiapoptotic proteins.64 In our 7670


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(12) Biswas, G.; Chatterjee, S.; Sarkar, S.; Acharya, K. Evaluation of Antioxidant and Nitric Oxide Synthase Activation Properties of Astraeus hygrometricus (Pers.) Morg. Int. J. Biomed. Pharma. Sci. 2010, 4, 21−26. (13) Lai, T. K.; Biswas, G.; Chatterjee, S.; Dutta, A.; Pal, C.; Banerji, J.; Bhuvanesh, N.; Reibenspies, J. H.; Acharya, K. Leishmanicidal and anticandidal activity of constituents of Indian edible mushroom Astraeus hygrometricus. Chem. Biodiversity 2012, 9 (8), 1517−1524. (14) Stanikunaite, R.; Radwan, M. M.; Trappe, J. M.; Fronczek, F.; Ross, S. A. Lanostane-Type Triterpenes from the Mushroom Astraeus pteridis with Antituberculosis Activity. J. Nat. Prod. 2008, 71, 2077− 2079. (15) Arpha, K.; Phosri, C.; Suwannasai, N.; Mongkolthanaru, W.; Sodngam, S. Astraodoric Acids A−D: New Lanostane Triterpenes from Edible Mushroom Astraeus odoratus and Their AntiMycobacterium tuberculosis H37Ra and Cytotoxic Activity. J. Agric. Food Chem. 2012, 60 (39), 9834−9841. (16) Biswas, G.; Rana, S.; Sarkar, S.; Acharya, K. Cardioprotective activity of ethanolic extract of Astraeus hygrometricus (Pers.) Morg. Pharmacologyonline 2011, 2, 808−817. (17) Biswas, G.; Acharya, K. Hypoglycemic Activity of Ethanolic Extract of Astraeus hygrometricus (Pers.) morg. in alloxan-induced diabetic mice. Int. J. Pharm. Pharm. Sci. 2013, 5, 391−394. (18) Biswas, G.; Sarkar, S.; Acharya, K. Free Radical Scavenging and Anti-Inflammatory Activities of the Extracts of Astraeus hygrometricus (Pers.) Morg. Lat. Am. J. Pharm. 2010, 29, 549−553. (19) Biswas, G.; Chatterjee, S.; Acharya, K. Chemopreventive activity of the ethanolic extract of Astraeus hygrometricus (Pers.) morg. on ehrlich’s ascites carcinoma cells. Dig. J. Nanomater. Biostruct. 2012, 7, 185−191. (20) Kingston, D. G. I. Modern Natural Products Drug Discovery and its Relevance to Biodiversity Conservation. J. Nat. Prod. 2011, 74 (3), 496−511. (21) Huang, Y. B.; He, L. Y.; Jiang, H. Y.; Chen, Y. X. Role of helicity on the cytotoxic mechanism of action of cationic-helical peptides. Int. J. Mol. Sci. 2012, 13 (6), 6849−6862. (22) Jorgensen, W.; Severance, D. L. Aromatic-Aromatic Interactions: Free Energy Profiles for the Benzene Dimer in Water, Chloroform, and Liquid Benzene. J. Am. Chem. Soc. 1990, 112 (12), 4768−4774. (23) Sheldrick, G. M. SHELXL97, Program for the Refinement of Crystal Structures; University of Gottingen: Gottingen, 1997. (24) Macrae, C. F.; Edington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; Van de Streck, J. Mercury: Visualization and Analysis of Crystal Structures. J. Appl. Crystallogr. 2006, 39, 453−457. (25) Brandenburg, K. DIAMOND; Crystal Impact GbR: Bonn, Germany, 1999. (26) Frisch, M.; Trucks, G. W.; Schlegel, H. B.; et al. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (27) Ahir, M.; Bhattacharya, S.; Karmakar, S.; Mukhopadhyay, A.; Mukherjee, S.; Ghosh, S.; Chattopadhyay, S.; Patra, P.; Adhikary, A. Tailored-CuO-nanowire decorated with folic acid mediated coupling of the mitochondrial-ROS generation and miR425-PTEN axis in furnishing potent anti-cancer activity in human triple negative breast carcinoma cells. Biomaterials 2016, 76, 115−132. (28) Bhattacharya, S.; Ahir, M.; Patra, P.; Mukherjee, S.; Ghosh, S.; Mazumdar, M.; Chattopadhyay, S.; Das, T.; Chattopadhyay, D.; Adhikary, A. PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a. Biomaterials 2015, 51, 91− 107. (29) Choi, J.; Albertin, F.; Klein, P.; Wang, Y.; Yip-Schneider, M.; Gage, E.; Wiesenauer, C.; Wiebke, E. A.; Lillemoe, K. D.; Schmidt, C. M. Doxorubicin’s effect on MEK activity predicts its chemotherapeutic response in hepatocellular carcinoma. J. Am. Coll. Surg. 2004, 199 (3), 80. (30) Buschauer, S.; Koch, A.; Wiggermann, P.; Mü ller, M.; Hellerbrand, C. Hepatocellular carcinoma cells surviving doxorubicin

in cancer treatment. Thereafter, further studies need to be carried on to unveil the effectiveness of astrakurkurol in defeating cancer both in vitro and in vivo. We enthusiastically presume that future study will validate the productiveness of astrakurkurol against various other cancers and may lead to its development as a promising cytotoxic agent for cancer treatment.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Arghya Adhikary: 0000-0002-7950-6527 Krishnendu Acharya: 0000-0003-1193-1823 Funding

This research was funded by the Department of Biotechnology, Government of West Bengal (Grant no. 1261 (sanc)/BT Estt./ RD-24/2013). S.N. acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India, for a CSIRUGC-NET Fellowship scheme. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the instrumental facilities provided by the Department of Botany (UGC-CAS Phase VI, VII), University of Calcutta, and Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, for use of the cell culture room facility and instrumental support. We would like to thank the reviewers for help in improving the quality of this manuscript.

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