Abstract
Two new scalarane sesterterpenoids, 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) and 12β-(3′β-hydroxypentanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (2), along with one known tetraprenyltoluquinol-related metabolite (3), were isolated from the sponge Carteriospongia sp. In leukemia Molt 4 cells, 1 at 0.0625 μg/mL (125 nM) triggered mitochondrial membrane potential (MMP) disruption and apoptosis showing more potent effect than 2 and 3. The isolates inhibited topoisomerase IIα expression. The apoptotic-inducing effect of 3 was supported by the in vivo experiment through suppressing the volume of xenograft tumor growth (47.58%) compared with the control. Compound 1 apoptotic mechanism of action in Molt 4 cells was further elucidated through inducing ROS generation, calcium release and ER stress. Using the molecular docking analysis, 1 exhibited more binding affinity to N-terminal ATP-binding pocket of Hsp90 protein than 17-AAG, a standard Hsp90 inhibitor. The expression of Hsp90 client proteins, Akt, p70S6k, NFκB, Raf-1, p-GSK3β, and XIAP, MDM 2 and Rb2, and CDK4 and Cyclin D3, HIF 1 and HSF1 were suppressed by the use of 1. However, the expression of Hsp70, acetylated tubulin, and activated caspase 3 were induced after 1 treatment. Our results suggested that the proapoptotic effect of the isolates is mediated through the inhibition of Hsp90 and topoisomerase activities.
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Introduction
Heat shock proteins (Hsp) comprise a certain group of highly conserved stress proteins which attracted attention due their overexpression in cancer tissues1,2. Overexpression of these proteins is related to metastatic potential, resistance to chemotherapy and poor prognosis3. They are named depending on their molecular weight (Hsp60, Hsp70 and Hsp90) and among the most studied protein is Hsp90. This protein is the most prominent member of the highly abundant chaperone proteins and it is essential for folding nascent polypeptide to control the activity, stability and protein sorting4. Hsp90 has been identified as a promising drug target for cancer treatment, because it can stabilize and activate a variety of survival proteins to maintain cancer phenotype and help cancer cells to overcome multiple environmental stresses5. There is considerable interest in develo** potential Hsp90 inhibitors, with a much simpler rationale, through the depletion of oncogenic Hsp90 clients6. The structure of this protein is composed of three major regions: an amino (N)-terminal domain with an adenosine triphosphate (ATP)-binding and hydrolyzing pocket (with ATPase activity), a middle domain involved in client protein recognition/binding, and a carboxy (C)-terminal domain7. Tanespimycin (17-allylamine-17-demethoxygeldanamycin, 17-AAG), the first Hsp90 inhibitor, was found to bind to the N-terminal regulatory pocket of Hsp90 and thus inhibiting its function. In Phase I clinical trials, it showed promising anticancer activity against multiple myeloma in combination with bortezomib8,9. However, further clinical development of 17-AAG was halted in 2010 because of poor solubility, limited bioavailability, unaccepted hepatotoxicity and the most important factor was the expiration of the patent in 20144,10,11. To date, several Hsp90 inhibitors have entered clinical trials, but none of them has been approved as an anticancer agent12.
Another important group of proteins, topoisomerases (Topo), has also attracted attention due to their crucial role in cell survival and replication13. Topoisomerases are classified into two main classes: topoisomerase I and topoisomerase II with each class performing specific functions in the cell. Topoisomerase II is composed of two isoforms, α and β, which share highly similar amino acid sequence (up to 70%)14. Topo IIα is essential for the survival of proliferating cells and can distinguish the handedness of DNA supercoils during relaxation reactions; however, Topo IIβ is dispensable at the cellular level15,16. Topo II disentangle topological problems, which regulate DNA replication, transcription and chromosome segregation, as well as processes related to tumorigenesis17. Inhibition of Topo II activity is one of the current therapeutic protocols targeting several cancers including lung, breast, lymphomas, testicular and sarcomas18.
The inhibition of Topo II activity is achieved either with poisons, which interfere with the topoisomerase-DNA complex or inhibitors, which suppress the catalytic turnover. Topo II poisons are divided into two distinct classes, interfacial poison and covalent poison based on their mechanism of action15,19. Anticancer drugs such as etoposide, doxorubicin, mitoxantrone and bioflavonoid analogs are considered interfacial poisons that bind non-covalently to the cleavage complex at the protein-DNA interface15,17. On the other hand, epigallocatechin galate (EGCG) and curcumin are covalent poisons which function distal to the active site of Topo II and increase the level of enzyme-mediated DNA cleavage by altering conformation of Topo II N-terminal protein gate20,21,22. Despite the effectiveness of Topo II poisons as anticancer drugs, they can trigger chromosomal breaks leading to secondary leukemogenesis14,23,24. One solution of this side effect was the development of catalytic Topo II inhibitors such as bisdioxopiperazines which eliminate the essential enzymatic activity of Topo IIα17. These compounds showed modest anticancer activity but did not induce secondary malignancies. Further developments of catalytic Topo II inhibitors can introduce new classes of highly effective and relatively safe anticancer agents.
Natural products scaffolds have played a crucial rule in guiding researchers to develop efficient anticancer agents targeting proteins necessary for cancer cell survival and replication11. Certain classes of secondary metabolites exhibited potent anticancer activity such as terpenoids, alkaloids, and flavonoids25. Terpenoids are the largest and most diverse group of secondary metabolites which are divided into several subgroups including monoterpenoids, diterpenoids, sesquiterpenoids, sesterterpenes, and triterpenoids26. Scalarane sesterterpenoids emerged as an interesting group of terpenoids which were isolated from marine sponges and shell-less mollusks. Scalarane-type sesterpenoids are 25C-terpenoids with tetra- or penta-cyclic skeletons derived from scalarin, which was firstly isolated from the sponge Cacospongia scalaris in 197227. Scalarane sesterterpenoids displayed a wide spectrum of interesting biological properties, such as antifeedant, antimicrobial, antifungal, ichthyotoxicity, antitubercular, antitumor, cytotoxicity, anti-HIV, antifouling, inhibition of platelet-aggregation, inhibition of transactivation for the nuclear hormone receptor (FXR, farnesoid X-activated receptor), stimulation of nerve growth factor synthesis, as well as anti-inflammatory activity28. Recent reports from our group revealed the potent cytotoxic potential of scalarane-type sesterterpenoids29,30. Carteriospongia (Phyllospongia) sp. sponge is a rich source of bioactive scalarane sesterterpenoids31. Unfortunately, many of the cytotoxic studies on sesterterpenoids evaluated their cytotoxic effect on cancer cell lines without deviling deeper into their mechanism of action32. Relieving the cytotoxic mechanism of action is crucial for discovering cytotoxic drug leads and fine tuning the active compounds to improve potency and reduce cytotoxicity. In the current investigation, we examined the content of EtOAc extract of Carteriospongia (Phyllospongia) sp. which led to the isolation of two new scalarane sesterterpenoids and one known tetraprenyltoluquinol-related metabolite. We evaluated the cytotoxic activity of isolates in vitro and in vivo along with their mechanism of action. The isolates caused disruption in mitochondrial membrane potential suggesting that the apoptotic effect is mediated through mitochondrial dysfunction. The cytotoxic effect of the new scalarane-type sesterterpenoid (1) on human leukemia Molt 4 cells involved induction of ER stress as demonstrated by the increase in ROS generation as well as ATF 6 cleavage and Chop expression. Compound 1 exhibited potent Hsp90 and Topoisomerase II (Topo II) catalytic inhibitory activity as determined by molecular docking and cell-free system. The treatment of Molt 4 cells with compound 1 suppressed client protein expression and accumulation of Hsp70 in the cytosolic compartment and well as reduced the transcription factors (HSF 1 and HIF 1) expression as demonstrated by Western blot and immunofluorescent analyses.
Results
Chemical identification of these marine terpenoids
The EtOAc extract of the freeze-dried specimen was fractionated by silica gel column chromatography and the eluted fractions were further separated utilizing normal phase HPLC to yield 1–3 (Fig. 1). The new compounds were named as 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) and 12β-(3′β-hydroxy-pentanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (2). The known compound was identified as 2-tetraprenil-1,4-benzochinone (3)33.
Compound 1 was obtained as colorless oil. The molecular formula of 1 was determined to be C31H48O6 by HR-ESI-MS (m/z 523.3397 [M+Na]+) and 13C NMR data (Table 1), implying eight degrees of unsaturation. IR absorptions were observed at 3448, 1717 and 1665 cm−1, suggesting the presence of hydroxyl, saturated carbonyl and α,β-unsaturated carbonyl functionalities. Resonances due to an aldehyde carbonyl carbon (δC 201.1), α,β-unsaturated carbonyl carbon (δC 198.2), ester carbonyl carbon (δC 172.1) and olefinic carbons (δC 142.9, CH; 138.4, C) in the 13C NMR and DEPT spectral data accounted for four double-bond equivalents, indicating a tetracyclic skeleton of 1. In the 1H NMR data, resonances of one olefinic proton (δH 7.06, s) and two oxygenated methines (δH 4.86, dd, J = 11.0, 4.5 Hz; 4.22, t, J = 9.0 Hz) were observed. The planar structure and all of the 1H and 13C chemical shifts of 1 were elucidated by 2D NMR spectroscopic analysis, in particular 1H-1H COSY and HMBC experiments (Fig. 2), suggesting a characteristic scalarin-type sesterterpenoid structure. Compound 1 was found to possess a 3-hydroxybutanoyloxy at C-12, a double bond at C-16/C-17, a ketone group at C-24 and an aldehyde group at C-25. Moreover, the structure of 1 was established unambiguously, and was found to be similar to that of a known compound, dendalone 3-hydroxybutyrate8,34. The relative configuration of 1 was suggested to be similar to that of dendalone 3-hydroxybutyrate by comparison of the chemical shifts and coupling constants of both compounds protons and was further confirmed by NOESY correlations (Fig. 2). The 3′R-configuration was determined by comparing the optical rotation (+10.7) with those of 2 (+5.7). The structure of 1 was thus found to possess the (4S*, 5S*, 8R*, 9R*, 10S*, 12R*, 13S*, 14S*, 18R*, 3′R*)-configuration.
Compound 2 molecular formula was deduced as C32H50O5 based on HR-ESI-MS (ion peak at m/z 537.3546 [M+Na]+) and 13C NMR data suggesting that 2 possesses one more CH2 group compared with 1. The NMR data of 2 (Table 1) showed similarity to those of 1 with the replacement of one 3-hydroxybutanoyloxy group at C-12 in 1 by one 3-hydropentanolyoxy group in 2. The structure of compound 2 was further confirmed by the COSY correlations from H2-29 to H3-32 and by the HMBC correlations from H-12 and H2-29 to C-28. Both compounds (1 and 2) were suggested to possess identical relative configuration based on the similarity in their NMR data. In order to identify the C-3′ configuration on the 3-hydroxybutanoyloxy group, the (S)- and (R)-MTPA ester derivatives, 2s and 2r, were synthesized with (−)-(R)-MTPA-Cl and (+)-(S)-MTPA-Cl, respectively. The Δδ-values indicated 3′R-configuration (Figure S19).
Apoptotic induction of these marine terpenoids via DNA damage and MMP dysfunction
The antiproliferative effect of the two new scalarane sesterterpenoids and the known tetraprenyltoluquinol-related metabolite was evaluated using MTT assay. Several cancer cell lines including leukemia K562, Molt 4, and HL 60 cells, prostate cancer LNCaP cells, colon cancer DLD-1 cells and breast cancer T-47D cells were used to evaluate the antiproliferative activity. Figure 3 summarized the data of IC50 values in cytotoxicity of these marine natural products against several cancer cell lines. Compound 1 exhibited the most potent cytotoxic activity with an IC50 of 0.01 μg/mL (2.08 nM) against all leukemia and one lymphoma cell lines. After 72 h treatment, the IC50 values of 1 against DLD-1, T-47D, LNCaP, Ca9-22 and Cal-27 cells were 2.33, 2.19, 13.87, 0.1, and 0.56 μg/mL, respectively. The IC50 values of 2 against leukemia K562, Molt 4 and HL 60 cells were 0.35 and 0.30, 0.22 μg/mL which were comparable to those of 3 (IC50 values: 0.70, 0.34 and 0.42 μg/mL). Molt 4 cell line was the most sensitive cell line as demonstrated by MTT assay. To detect whether the cytotoxic effect of these terpenoids was associated with the mitochondria-related apoptosis, we assessed the population of apoptosis and disruption of mitochondrial membrane potential in Molt 4 cells with annexin-V/PI and JC-1 staining. After 24 h, compounds 1, 2 and 3 resulted in a dose-dependent (0, 0.0625, 0.125 and 0.25 μg/mL) increase in the apoptotic population of Molt 4 cells and disruption in mitochondrial membrane potential (Fig. 4A,B). These results proved that the cytotoxic activity of these marine terpenoids might be mediated through mitochondrial dysfunction leading to induction of apoptosis.
The ability of Topo II to generate DNA damage in the presence of Topo II-targeting agents led to the hypothesis that an important determinant of drug sensitivity was related to the overall level of Topo II17. In our previous report, 10-acetylirciformonin B, a marine sesterterpenoid analog, significantly inhibited Topo IIα activity35. Such findings encouraged us to evaluate the effect of the isolates on Topo II activity. In order to confirm whether the DNA damage-induced by the isolated sesterterpenoids involved the inhibition of Topo II activity, a cell-free DNA cleavage assay using an enzyme-mediated negatively supercoiled pHOT1 plasmid DNA was applied. A linear DNA strand was observed upon treating the supercoiled pHOT1 plasmid DNA with etoposide, a standard topo II poison (Lane 16). The use of compound 1 in increasing concentrations (0.08, 0.312, 1.25, 5, and 20 μg/mL) significantly inhibited DNA relaxation by 12, 13, 17, 20 and 99%, respectively, compared with the control supercoiled DNA and resulted in the formation of supercoiled DNA products in the presence of topo IIα (Lanes 6–10). Additionally, 2 and 3 significantly inhibited DNA relaxation by 12, 23, 36, 90 and 99% (Lanes 11–15); and 7, 13, 28, 90 and 98% (Lanes 1–5), respectively. These marine terpenoids suppressed Topo II activity, resulting in the inhibition of supercoiled DNA relaxation in a dose-dependent manner (Fig. 5A). Furthermore, 1-3 inhibited topo II activities with IC50 of 1.98, 0.37 and 0.43 μg/mL, respectively as demonstrated by the cell-free system. To determine whether the apoptotic mechanism of these compounds affects γH2AX (as a biomarker of DNA damage) induction, Western blot analysis was employed to examine the activation of γH2AX. Compounds 1 and 2 significantly enhanced the expression of γH2AX in a dose-dependent manner, but the activation of DNA damage was not observed on 3 (Fig. 5B). These results suggested that these compounds could act as potent catalytic inhibitors of Topo IIα.
2-Tetraprenil-1,4-benzochinone (3) inhibited tumor growth in in vivo human Molt 4 tumor xenograft animal model
Compounds 1 and 2 induced apoptosis in several cancer cell lines; however, the amounts isolated were insufficient for further examination of their in vivo antitumor effect. On the other hand, the amount separated from 3 was sufficient to study the in vivo anti-tumor activity by evaluating its effect on tumor growth of human leukemia Molt 4 in xenograft animal model. Molt 4 (1 × 106) cells were inoculated subcutaneously at the right flank of female immunodeficient athymic mice. After 33-day of treatment, the tumor growth of Molt 4 cells was significantly suppressed under the influence of 3 (1.14 μg/g) intraperitoneal injection. The average tumor size on day 33 in the control group was 404.63 mm3, whereas the average tumor size in compound 3-treated group was 212.10 mm3 (Fig. 6A). The tumor size was significantly lower in compound 3-treated group as compared to the control group (p < 0.05) with no significant difference in the mice body weights (Fig. 6B). These results suggested that 3 exhibited anti-tumorigenic effect in vivo xenograft model.
Apoptotic effect of 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) involves the induction of ROS generation, ER stress and DNA damage in Molt 4 cells
Oxidative stress is manifested by ROS overexpression and cannot be balanced by the available antioxidant machinery36. Compound 1 can damage the integrity of mitochondria, which are the major production sites of the superoxide anion, ozone37. The induction of the intracellular formation of ROS by 1 was determined with a carboxyl derivative of fluorescein, carboxy-H2DCFDA dye using flow cytometric analysis38. As shown in Fig. 7A, treatment with 1 (0.0625 μg/mL) for 0.5, 1, 2 and 3 h resulted in 2.09-, 1.51-, 1.06- and 1.01-fold increase in ROS levels, respectively, as compared with the mean fluorescence index (MFI) of the control. In addition, ROS generation could induce ER stress leading to mitochondria-related apoptosis39,40. To further investigate if ER stress is involved in the apoptotic effect induced by 1, Western blot analysis was used to determine the expression of ER stress-related proteins. In a time-dependent manner, 1 promoted the levels of Bip, Chop, Grp 94 as well as the activation and cleavage of ATF 6 but suppressed the levels of PERK and IRE 1 (Fig. 7B). Furthermore, the effect of 1 on the release of intracellular Ca2+, was evaluated using fluorescent calcium indicator, Fluo 3. The flow cytometric results showed that the treatment with 1 at different time intervals (0.5, 1, 2, 3, 6 and 18 h) induced 1.03-, 1.05-, 1.06-, 1.37-, 2.41-, and 1.06-folds increase in the intracellular Ca2+ accumulation, respectively as compared to MFI of the control (Fig. 7C), indicating that ER stress was induced by compound 1 following the redox stress. Moreover, we noticed a significant increase of PARP, caspases-8 and −9 cleavages in a time-dependent manner. The induction of the typical executor caspases-3 and −7, as well as γH2AX, was also observed using Western blot analysis (Fig. 7D). To confirm the induction of DNA damage by 1 in Molt 4 cells, a comet assay under neutral electrophoresis condition was utilized. The effect of 1 (0.0625 μg/mL) at different time intervals (0, 3, 6 and 9 h) on the level of nuclear DNA integrity was determined. As shown in Fig. 7E 1 increased the degree of DNA migration in Molt 4 cells. The DNA migration was represented by the induction of DSBs in a time-dependent increase, as indicated by the abnormal tails’ sizes in the comet assay.
12β-(3′β-Hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) as a potent inhibitor of Hsp90
It is known that mitochondria are critical intracellular loci of ROS production and ROS exposure can lead to the mPTP opening41. The antioxidant, microsomal GST1, in the inner mitochondrial membrane can interact with the mitochondrial permeability transition (MPT) regulator proteins, such as ANT and/or CypD, to form a MPT pore contributing to mitochondria-mediated cell death42. It was reported that mitochondrial homeostasis of tumor cells is regulated by an organelle-specific Hsp90 chaperone network43. To investigate whether the inhibition of Hsp90 participates in the apoptosis induced by 1, we performed molecular docking of Hsp90 with the available crystal structure to gain insight of 1 binding mode. Our studies revealed that 1 could be docked to the N-terminal domain of Hsp90 with the binding energy of −10.93 Kcal/mol over the first Hsp90 N-terminal inhibitor, 17-AAG (−6.63 Kcal/mol), presumably because 1 formed non-classic hydrogen bonds with GLY132 and GLY135 at a distance of 3.27 and 3.66 Å; the classic hydrogen bonds with residues LYS112 (1.92 Å), PHE138 (2.31 Å and 2.45 Å) and ASN106 (2.45 Å); the hydrophobic bonds with residues ALA 55 (3.63 and 3.69 Å) and MET 98 (3.85 and 4.96 Å) (Fig. 8A). According to the molecular docking experiments, compound 1 inhibits Hsp90 with an inhibition constant of 9.67 nM (>1430 folds) which was more potent than 17-AAG (10.83 μM). To fully understand the differences in activity between 1 and 17-AAG, we further examined the antiproliferative activity of 17-AAG in Molt 4 cells by MTT assay. Treatment of Molt 4 with increasing concentrations (0, 0.4, 2 and 10 μM of 17-AAG for 24 h induced a suppression in cell growth with IC50 values of 4.2 μg/mL (7.2 μM). (Fig. 8B). According to the MTT results using Molt 4 cells, the antiproliferative activity of 1 (0.01 μg/mL, 19.1 nM) was 377 folds higher than that of 17-AAG.
Recent reports indicated that Hsp90 inhibitors induced HSF 1-dependent expression of Hsp7044, and the genetic deletion of HSF 1 reduced the association of Hsp90 with its kinase client proteins45,73. The gels were directly scanned with image analyzer, and the area representing supercoiled DNA calculated to evaluate the concentration that the compounds caused 50% inhibition (IC50) of Topo II activity.
Neutral Comet Assay for Detection of DNA Double-strand Breaks (DSBs)
The assay was carried out using a CometAssayTM Kit (Trevigen, Gaithersburg, MD, USA) following the manufacturer’s protocol for the neutral Comet assay. Briefly, cancer cells (2 × 105 cells/mL) were treated with compound 1 (0.0625 μg/mL) at the indicated time. Cells were combined with 1% low melting point agarose at a ratio of 1:10 (v/v) and immediately 75 μL of the mixture was pipetted onto CometSlideTM and allowed to set at 4 °C in the dark. The slides were immersed in ice-cold lysis solution (Trevigen) for 30 to 60 min. The slides were placed in a horizontal electrophoresis apparatus and electrophoresed in 1X TBE (90 mM Tris-HCl, 90 mM boric acid, and 2 mM EDTA, pH 8.0) at 20 V for 10 min. The samples were then fixed in 70% ethanol and dried before stained with 1:10,000 SYBR Green I (Trevigen) to visualize cellular DNA. The fluorescence images were analyzed using the TriTek Comet Image program to circumscribe the “head” and the “tail” regions of each comet and the integrated fluorescence values of each defined area were recorded. The comet length was measured from the trailing edge of the nucleus to the leading edge of the tail. This length was indicative of the extent of DNA damage. Calculations were averaged per replicate.
Western Blotting Analysis
Cell lysates were prepared by treating the cells for 30 min in RIPA lysis buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 100 μg/mL phenylmethylsulfonyl fluoride and 30 μg/mL aprotinin) (all chemicals were from Sigma)38. The lysates were centrifuged at 20,000 × g for 30 min, and the protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce). Equal amounts of proteins were respectively separated by 7.5%, 10% or 12% of SDS-polyacrylamide gel electrophoresis and then were electrotransferred to a PVDF membrane. The membrane was blocked with a solution containing 5% non-fat dry milk TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% Tween 20) for 1 h and washed with TBST buffer. The protein expressions were monitored by immunoblotting using specific antibodies. These proteins were detected by an enhanced chemiluminescence kit (Pierce).
Immunofluorescence Analysis
After treatment with the tested compound, cells were fixed with 4% paraformaldehyde in 50 mM HEPES buffer (pH 7.3) for 30 min, and permeabilized for 20 min with 0.2% Trition X-100 in PBS (pH 7.4). To prevent non-specific protein binding, cells were incubated with 5% BSA in PBS containing 0.05% Trition X-100 (T-PBS) for 1 h at room temperature. Cells were then incubated with the primary Hsp70 antibodies (1: 500) for 2 h and further with secondary antibodies (Alexa Fluor 586-conjugated goat anti-mouse IgG (H + L) (Life Technologies, Carlsbad, CA, USA) diluted at 1:1000 for 1 h at room temperature. After washing with PBS, cells were observed under a FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Human Leukemia Molt 4 Cells Xenograft Animal Model
Establishment of nude mice with xenografts was performed as described previously38. Six-week-old male immunodeficient athymic mice were purchased from the National Laboratory Animal and Research Center (Taipei, Taiwan). All of the animals were maintained under standard laboratory conditions (temperature 24–26 °C, 12–12 h dark-light circle) and fed with laboratory diet and water. This study was approved by the Animal Care and Treatment Committee of Kaohsiung Medical University (IACUC Permit Number 101136). All experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and all efforts were made to minimize animal stress/distress. Molt 4 cells (1 × 106) resuspended in 0.2 mL PBS were injected s.c. into the right flank of each mouse, and tumor growth was monitored every day. Fourteen days after tumor cell injection, mice with confirmed tumor growth were randomly divided into two groups. Compound 3 (1.14 μg/g) was intraperitoneally administered to the treatment group, and the control group received solvent only. Compound 3 was administrated every other day for 33 days. Animals were sacrificed by carbon dioxide. Tumor size was measured three times a week using calipers and tumor volumes were calculated according to the standard formula: width2 × length/2.
Molecular Modeling Assay
The molecular docking was performed by Autodock 4.2 with Lamarckian Genetic Algorithm74. The target macromolecule, Hsp90 protein (PDB ID: 1YET), was obtained from the Protein data bank (http://www.rcsb.org/pdb/home/home.do)75. The co-crystalized protein substrates, including ligands, water and small molecules were removed, and the Polar hydrogens and Kallman united atom charges were added to the protein for docking calculation by AutoDock Tool 1.5.4 interfaces (ADT)76. The ligands were optimized with MMFF94 force field by ChemBio3D software (version 11.0; Cambridge Soft Corp.). Polar hydrogens and Gasteiger charges were also added to the ligand for docking study by ADT. The Grid box calculated by AutoGrid program was centered at the activity site of Hsp90 with dimensions 56 × 56 × 56 Å grid points at spacing of 0.375Å and its size is big enough to allow the ligand move freely in the search space. All docking parameters were set to default except for the following parameter: maximum number of energy evaluation increase to 25,000,000 per run. The docking results were analyzed by ADT and shown by Accelrys Discovery Studio v3.5 client software (Accelrys Inc, San Diego, CA (2005)).
Statistics
The results were expressed as mean ± standard deviation (SD). Comparison in each experiment was performed using an unpaired Student’s t-test and a p value of less than 0.05 was considered to be statistically significant.
Additional Information
How to cite this article: Lai, K.-H. et al. Antileukemic Scalarane Sesterterpenoids and Meroditerpenoid from Carteriospongia (Phyllospongia) sp., Induce Apoptosis via Dual Inhibitory Effects on Topoisomerase II and Hsp90. Sci. Rep. 6, 36170; doi: 10.1038/srep36170 (2016).
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Acknowledgements
This research was supported by grants from the National Museum of Marine Biology & Aquarium as well as the Ministry of Science and Technology (MOST 103-2320-B-291-001-MY3 and MOST 104-2320-B-259-003-MY3), Taiwan, awarded to J.-H. Su and M.-J. Lu, respectively. We are grateful to the National Center for High-Performance Computing for computer time and facilities. We also thank the Center for Resources, Research, and Development of Kaohsiung Medical University for the ChemBioOffice technical support.
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Design of the experiments: M.-C.L., J.-H.S., K.-H.L. and Y.-C.L. Execution and analysis of the phytochemical experiments: J.-H.S., K.-H.L., M.-K.W., Y.-C.D., Y.-M.H. and M.E. Execution of the pharmacologic experiments: M.-C.L., Y.-C.L., C.-F.W., C.-H.C., J.-C.Y. and G.-Y.C. Contribution in reagents/materials/analysis tools: M.-C.L., J.-H.S. and Y.-C.L. Writing and revising the manuscript: M.-C.L., K.-H.L., M.E., J.-H.S. and Y.-C.D. All authors reviewed the manuscript.
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Lai, KH., Liu, YC., Su, JH. et al. Antileukemic Scalarane Sesterterpenoids and Meroditerpenoid from Carteriospongia (Phyllospongia) sp., Induce Apoptosis via Dual Inhibitory Effects on Topoisomerase II and Hsp90. Sci Rep 6, 36170 (2016). https://doi.org/10.1038/srep36170
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DOI: https://doi.org/10.1038/srep36170
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