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Probing Inhibitory Effects of Antrodia camphorata Isolates Using Insect Cell-Based Impedance Spectroscopy: Inhibition vs Chemical Structure Keith B. Male,† Yerra Koteswara Rao,‡ Yew-Min Tzeng,*,‡ Johnny Montes,† Amine Kamen,† and John H. T. Luong*,†,§ Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2, Institute of Biotechnology, Chaoyang UniVersity of Technology, Wufeng, 41349 Taiwan, Republic of China, and Department of Chemistry, UniVersity College Cork, Cork, Ireland ReceiVed June 4, 2008
A continuous online technique based on electric cell-substrate impedance sensing (ECIS) was used for probing inhibitory effects on Spodoptera frugiperda Sf9 insect cells exposed to structurally similar compounds isolated and purified from the fruiting bodies of the fungus Antrodia camphorata. Such chemicals consisted of three ergostane-related steroids and five lanosta-related triterpenes, which are known for their diverse properties and use in the formulation of nutraceuticals and functional foods. The half-inhibition concentration (ECIS50), the level at which 50% inhibition of the resistance response was obtained, was determined from the response function to establish inhibitory effects of the different isolates. A slight change in their chemical structures resulted in significant effects on inhibition as probed by impedance spectroscopy. The ergostane-related steroids were mostly inhibitory, but replacing their ketone groups with hydrogen or hydroxyl groups significantly reduced the inhibition. Similarly, the addition of methyl or carboxymethyl groups also lowered the inhibition. Removal of the double bond conjugation within the rings (sulfurenic acid) of the isolate drastically reduced the inhibition. Introduction Antrodia camphorata (syn. Taiwanofungus camphoratus), a Ganoderma-like fungus, belongs to the Polyporaceae, Basidiomycotine family and grows in a unique host, the endemic perennial tree Cinnamomun kanehirai (Bull camphor tree), at an altitude of 450-2000 m in the mountains of Taiwan. As an indigenous and rare species in Taiwan, it has been known as “niu-chang-chih” or “niu-chang-ku”. A. camphorata has not only long been utilized to treat a wide variety of diseases but has also recently drawn the attention of the pharmaceutical industry. Traditionally, it has been used in the treatment of diverse conditions such as abdominal pain, diarrhea, drug intoxication, hypertension, and skin itching as well as to improve the immune system and liver function (1). Several biological activities have been reported for A. camphorata, including anticancer (2-5), antihepatotoxic (6, 7), antihypertensive (8), anti-inflammatory (9, 10), antioxidant (11), and neuroprotective activities (12). A. camphorata inhibits LPS/IFN-γ induced inflammatory mediator production in macrophages, as well as tumor cell proliferation (13). Previous phytochemical investigations on A. camphorata have resulted in the isolation of diterpenoids, triterpenoids, sesquiterpene lactone, benzenoids, and polysaccharides (10, 12, 14-20). However, only limited work has been carried out on the constituents of this fungus and their biological activities. Commercially available A. camphorata is used in the formulation of nutraceuticals and functional foods in Taiwan. Hence, it would be * To whom corresponding should be addressed. E-mail: (Y.-M.T.)
[email protected] and (J.H.T.L.)
[email protected] or
[email protected]. † National Research Council Canada. ‡ Chaoyang University of Technology. § University College Cork.
worthwhile to fully characterize its constituents, therapeutic activities, and possible cytotoxicity. Cell spreading, morphology, and micromotion, three important parameters in tissue culture, have been quantified using an electrical method referred to as electric cell-substrate impedance sensing (ECIS) (21, 22). In brief, cells drift downward through the culture medium and settle on the bottom of tissue culture wells (volume of ∼9 ( 9 × 10 mm3), each containing a 250 µm diameter circular gold electrode. The cells then begin to attach and spread on the electrode surface, precoated with a binding protein. Eventually, the confluent cell layer formed affects the current flow since these adhered cells will act as insulating particles due to their plasma membrane. The measured impedance changes drastically after the cell attachment and spreading, due to an interference with the free space above the electrode. The changing impedance can be continuously monitored and interpreted to reveal information about cell spreading (23, 24). The impedance, a result of the coordination of many biochemical reactions, is very sensitive to operating conditions such as pH, temperature, and chemical compounds added to the culture medium. The broad response to changes in the environment allows this method to serve as a general tool for probing cell spreading and motility as well as an alternative to animal testing for toxicology studies. The applicability of ECIS for inhibition assays has been demonstrated using toxic or noxious agents such as cytochalasin D, a cytoskeletal inhibitor (25), prostaglandin E2 (inflammatory mediator) (26), bacterial protease, bacterial proteins that perturb extracellular matrix and cytoskeleton (27), environmental toxins such as heavy metals and nitrotoluenes (28-30), as well as nanomaterials including quantum dots and gold nanoparticles (31). To date, mammalian
10.1021/tx800202a CCC: $40.75 2008 American Chemical Society Published on Web 09/25/2008
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Figure 1. Chemical structures of the eight isolates from A. camphorata.
cells have been used extensively with ECIS to probe cell behavior including spreading, micromotion, and cytotoxicity (29-31). This paper describes an online technique based on ECIS for the continuous assessment of the behavior of Spodoptera frugiperda Sf 9 insect cells exposed to the eight isolates from A. camphorata (Figure 1). The attached and spread S. frugiperda Sf 9 insect cells will result in a significant change in the measured impedance due to cell attachment and spreading. Exposure of S. frugiperda Sf9 insect cells to these isolates, if cytotoxic or inhibitory at the substratum level, will lead to alterations in cell behavior, and the resulting chemical effect can be screened by measuring the impedance change. The insect cell-baculovirus host vector system has been increasingly utilized for the expression of heterologous proteins, including many high-value biologicals such as growth factors, hormones, and vaccines for both human beings and animals (32, 33).
Experimental Procedures Materials. All materials were obtained commercially (guaranteed reagent grade) and used without further purification. Silica gel for column chromatography (CC) (0.063-0.200 mm) was a product of Merck Co. Thin-layer chromatography (TLC) was performed on Merck TLC plates (0.23 mm thickness), with compounds visualized by spraying with 8% (v/v) H2SO4 in ethanol and then heating on a hot plate. NMR (nuclear magnetic resonance) spectra were measured on a Varian Unity Inova-600 VXR-300/51 spectrometer, using trimethylsilane (TMS) as an internal standard. ESIMS (electrospray ionization-mass spectrometry) spectra were recorded on a Thermo-Finnigan LCQ Advantage system. The fruiting bodies of A. camphorata were obtained through a solid state cultivation (SSC) process (accession numbers G908AC). The freshly collected materials were dried under shade, sliced into small pieces, pulverized using a mechanical grinder, passed through 40 mesh sieve, and preserved in an airtight container for further use. The voucher specimens were deposited in the Herbarium of
Inhibitory Effects of A. camphorata Isolates
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Table 1. Absorption Coefficients in Ethanol of 100 µM A. camphorata Isolates (1-8) compound
UV absorption (nm)
absorption coefficient ()
1 2
267 242 235 243 235 243 235 267 251 243 270 254
10232 8150 8108 13680 12920 14820 14189 14075 11486 14126 11117 9223
3 4 5 6 7 8
the Institute of Biotechnology, Chaoyang University of Technology, Taiwan. Concanavalin A (Con A, type III) from CanaValin ensiformis and Trypan Blue were purchased from Sigma-Aldrich (St. Louis, MO). Extraction and Isolation of Isolates from A. camphorate. The powdered fruiting bodies of A. camphorata (30 g) were extracted with CHCl3 (5 × 200 mL) and MeOH (5 × 200 mL), successively using a Soxhlet extractor until the refluxed solvent became colorless. After extensive extraction, all of the extracts were filtered individually, and the solvent was dried by rotary evaporation under reduced pressure at a temperature maximum of 35 °C to give brownishblack colored residues in yields of 12.0 (40.0%, w/w) and 1.78 g (5.9%, w/w), respectively. A part of the residue from the chloroform extract (1 g) was preserved, and the remaining (11 g) was subjected to silica gel chromatography (4 cm × 90 cm, 0.063-0.200 mesh). The column was eluted with solvents of increasing polarity using a mixture of n-hexane/EtOAc. The eluates were collected in fractions of 200 mL each and concentrated. Following the TLC analysis, eluates of similar profiles were combined to give five fractions (A-E), which were processed by chromatography and eluted with a mixture of n-hexane/EtOAc. Fraction B (2.7 g) was processed using silica gel CC eluting with a gradient of n-hexane/ EtOAc to give methyl antcinate B or zhankuic acid A methyl ester (1, 27 mg, 0.09%, w/w), dehydroeburicoic acid or 24-methylenelanosta-7,9-(11)-diene-3β-ol-21-oic acid (2, 170 mg, 0.56%, w/w), and 15R-acetyl dehydrosulphurenic acid or 15R-acetoxy-24-methylenelanosta-7,9-(11)-diene-3β-ol-21-oic acid (3, 95 mg, 0.32%, w/w). Fraction C (3.0 g) was further separated using a silica gel column eluting with n-hexane/EtOAc to afford 3β,15R-dihydroxy lanosta-7,9-(11), 24-triene-21-oic acid (4, 60 mg, 0.2%, w/w) and antcin B, zhankuic acid A, or 4R-metylergosta-8,24 (28)-diene3,7,11-trion-26-oic acid (5, 350 mg, 1.16%, w/w), respectively. Fraction D (4.5 g) was further separated using a silica gel column eluting with a gradient of n-hexane/EtOAc, resulting in five subfractions. Dehydrosulfurenic acid or 24-methylenelanosta-7,9(11)-diene-3β,15R-diol-21-oic acid (6, 470 mg, 1.56%, w/w) and antcin H, zhankuic acid C, or 3R,12R-dihydroxy-4R-methylergosta8,24 (28)-diene-7,11-dione-26-oic acid (7, 65 mg, 0.21%, w/w) were obtained from subfractions D-3 and D-4, respectively. Sulfurenic acid or 24-methylenelanosta-8-ene-3β, 15R-diol-21-oic acid (8, 75 mg, 0.25%, w/w) was obtained from fractions E (1.2 g). All of the constituents were found (by TLC) to be present in the original extract of the A. camphorata material. The structures of compounds 1-8 (Figure 1) were determined by 1H and 13C NMR spectroscopy and by comparison of the spectral data with those of published values (Supporting Information). UV Absorption. All UV/visible absorption spectra for A. camphorata isolates (1-8) solubilized in ethanol were obtained at room temperature on a Varian Cary Win UV-50 spectrophotometer. Test solutions (100 µM) were prepared by diluting known amounts of a given stock solution with ethanol. A 1 cm path length Suprasil cuvette was used in all experiments. Baseline corrections were also conducted prior to each measurement. The molar absorption coefficients and the UV-vis spectra of the eight isolates are given in Table 1 and Figure S1, respectively (Supporting Information).
Cell Line and Culture Conditions. S. frugiperda Sf9 cells were maintained in 125 mL disposable Erlenmeyer flasks with a working volume of 20 mL in serum-free SF-900 II medium (Gibco BRL, Canadian Life Technologies, Burlington, ON, Canada). Cells were cultured weekly at 0.4 × 106 cells/mL at 27 °C, pH 6.2, with agitation at 110 rpm. The monitoring of the cell count and viability by the Trypan Blue exclusion assay during the growth was performed with a CEDEX Innovatis cell counter (Bielefeld, Germany). Sf9 cells, inoculated at an initial cell density of 0.4 × 106 cells/mL, were grown to the midexponential phase 2.5-3 × 106 cells/mL, and the resulting cells were aseptically centrifuged at 1500 rpm for 4 min. Pellets were thereafter suspended at a cell concentration of 3 × 106 cells/mL in a fresh medium. Electrode Coating and Cell Addition. Concanavalin A (Con A, 0.40 mL, 0.5 mg/mL, prepared fresh daily in 50 mM PBS, pH 7.4, with the aid of sonication for 1 h) was added into each of the eight wells of a sensing chip (8W1E, Applied Biophysics, Troy, NY) to coat the detecting gold electrodes as previously described by Luong et al. (28). Con A binds quickly (90% of the change occurs in the first 10 min) to the electrode surface as confirmed by an impedance increase from 9290 ( 218 to 11442 ( 295 Ω, corresponding to a capacitance decrease from 4.30 ( 0.13 to 3.48 ( 0.08 nF (95% confidence interval, n ) 4). However, there was no appreciable change in the resistance. The attachment of Con A to the gold electrode was very stable as reflected by stable and constant impedance reading over 24 h. After protein adsorption (∼30-60 min), the wells were washed three times with 0.85% NaCl, and 0.4 mL of culture medium was placed in each well. The impedance baseline was monitored for 1-2 h at 27 °C in a humidified chamber with the ECIS impedance system. The wells were then emptied, and 0.4 mL of cell suspension (∼3 × 106 cells/ mL) was added into each well. Isolate samples (2-3 mg) from A. camphorata were dissolved in ethanol (∼0.5 mL) with the aid of sonication to concentrations of ∼10 mM. The exact concentration was determined from the molar absorption coefficients as shown in Table 1. The isolate samples (30 µL) were added to cell suspensions (1.5 mL at 3 × 106 cells/mL) at various concentrations before adding 0.4 mL (from the same isolate sample-cell suspension mixture) to two or three wells to test for possible inhibitory effects. For each isolate, six concentrations including a control with 30 µL of ethanol were tested at the same time, and each isolate was analyzed 2-3 different times. Impedance Measurement with ECIS. Detailed information on ECIS impedance measurement has been reported elsewhere (28-31). In brief, the system can measure up to 16 sample wells (two chips of eight wells, each containing a singly addressable detecting electrode) per experiment. A common counter gold electrode is shared by the eight detecting electrodes, and the two electrodes (detecting gold electrode and counter gold electrode) of the well are connected to a lock-in amplifier of the ECIS system. Cell behavior should not be affected if the applied potential is 1 V AC or less (34, 35). The impedance of each well was measured every 2 min at 4 kHz, and the system acquired resistance, impedance, and capacitance data. However, as larger changes occurred in the resistance, we have focused on these changes in this study. The ECIS50 value derived from the time response function, f(C, t), was calculated as described by Xiao et al. (29). For simplification of plots and calculations, data points at 30 min intervals were selected from the raw resistance data. The sensing chip could be temporarily removed (pause function from the software) from the ECIS system incubator and placed on a Wilovert AFL 30 inverted microscope (Hund, Germany) equipped with a digital video camera (KP-D50U, Hitachi, Tokyo, Japan) to observe the cells during experimentation.
Results and Discussion Response of Sf9 Insect Cells. The gold electrode was fully covered by Con A, a lectin purified from ConcanaValis ensiformis. This study confirmed that Con A provoked the best adhesion behavior for this insect cell line as reported previously (28). This was an interesting observation since most mammalian
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Figure 2. Resistance response (Ω) of Sf9 insect cells to various concentrations (µM) of A. camphorata isolate 5: (a) 0, (b) 1.2, (c) 2.3, (d) 4.6, (e) 11.6, and (f) 23.2.
cells require a protein such as fibronectin, collagen, laminin, etc. to promote cell adhesion and spreading. The resistance of the culture medium without cells was ∼2.5 kΩ, while with cells there was a slight increase to ∼3.0 kΩ. Without the A. camphorata isolates, the cells descended to the bottom of the well within 20 min as observed by the video-enhanced microscope, and as they spread, the cells changed from round shapes to flattened forms with much larger dimensions. Cell-substratum (cell-Con A) interactions including spreading, morphology, and cell motility require a complex series of events to occur in a regulated and integrated fashion. As the cells spread, they alter the effective area available for current flow, causing a significant increase in the resistance of the well by ∼7.2 to 10.2 kΩ as shown in Figure 2 (curve a). The number of normal Sf9 cells to completely cover an 8W1E detecting electrode coated with Con A was previously reported (28) to be between 150 and 200 cells; therefore, the estimated resistance change contributed by each attached cell was about 35-50 Ω/cell. The addition of a low concentration (1.2 µM) of isolate 5 (Figure 2, curve b) to the cell suspension had little effect on the resistance signal. However, as the concentration was increased (2.3-23.2 µM), the resistance change was significantly decreased (Figure 2, curves c-f). Inverted fluorescent microscopy (Figure 3a,b) confirmed that the insect cells in the absence of the isolate were intact and well spread on the Con A-coated electrode surface even after washing the wells with saline three times. However, insect cells exposed to isolate 5 (23.2 µM) for 24 h were more spherical (Figure 3c,d) as compared to the control cells. Extensive washing of the ECIS wells with saline removed the effector cells from the electrode indicating that they were no longer firmly attached to Con A precoated on the gold surface. Isolate 5 could bind directly to either bare gold surfaces or Con A-coated gold surfaces as illustrated by an impedance increase in both cases. Without Con A coating, however, the impedance gradually dropped over 24 h, implying instability of the isolate-protein linkage as compared to the Au-S linkage between Con A and gold. The presence of the isolate (up to 240 µM) bound to the Con A-coated electrode surface did not interfere with the resistance response to insect cells as the resistance response obtained with or without isolate was virtually comparable. The wells were extensively washed to remove excess isolate before adding the cells. Half-Inhibition Concentration (ECIS50) for A. camphorata Isolate 5. For the effector cells, the resistance change (∆Rs) of the well is dependent on the number (No) of initial cells attached on the detecting electrode, the toxicant concentration
Male et al.
(C), and the exposure time (t) as reported by Xiao et al. (29). The resistance change normalized by No is defined as the cell response to the toxicant measured by ECIS, f(C, t) ) ∆Rs/No. As a control with no toxicant, C is equal to zero and f(0, t) increases as the cells spread on the electrode and reaches a plateau. In the presence of toxicant, f(C, t), after an initial increase, the value decreases and can even approach zero, indicating total cell death at high toxicant concentrations. The inhibitor concentration required to achieve 50% inhibition of the response is defined as the half-inhibition concentration (ECIS50) or f (ECIS50, t)/f (0, t) ) 50%. The ECIS50 for isolate 5 was calculated from the data obtained in Figure 2. The time-response function f(C, t), was used to construct a series of inhibition curves at any given time t0 (>2.5 h) for the series of isolate concentrations used in Figure 2. The insect cells settled to the electrode surface coated with Con A very rapidly (20 min) as confirmed by inverted fluorescent microscopy. Very few cells (
