Biotechnol Lett (2007) 29:1307–1313 DOI 10.1007/s10529-007-9414-6

ORIGINAL RESEARCH PAPER

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip Chengjun Huang Æ Ailiang Chen Æ Min Guo Æ Jun Yu

Received: 29 August 2006 / Revised: 10 April 2007 / Accepted: 17 April 2007 / Published online: 26 June 2007
Springer Science+Business Media B.V. 2007

Abstract A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 ± 0.9 mF/cm2 to 6.4 ± 0.6 mF/cm2 after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives

Chengjun Huang and Ailiang Chen contributed equally to this work. C. Huang
J. Yu (&) Department of Electronic Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei Province 430074, China e-mail: [email protected]; [email protected] A. Chen Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China A. Chen Medical Systems Biology Research Center, Tsinghua University, School of Medicine, Beijing 100084, China C. Huang
A. Chen
M. Guo
J. Yu National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Chang Ping District, Beijing 102206, China

a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents. Keywords Apoptosis
Electrorotation
Membrane capacitance
Membrane conductance

Introduction Apoptosis is studied both as a target for drug discovery and as a cellular process. The majority of existing techniques to monitor apoptosis in vitro is based on fluorescence probes designed to specifically label relevant molecules in apoptotic cells or by flow cytometry (Rodriguez and Schaper 2005). Other more biochemical assays read the activity of apoptosis-related enzymes or determine the degree of DNA fragmentation (McConkey 1998). Each of these methods detects different morphological or biochemical features. At the same time, the vast history of experience of traditional Chinese medicine (TCM) may facilitate the identification of novel anti-leukemic compounds. Bufalin is one of the major active components prepared from toad (Chan Su), venom extracts which is used as a TCM. Bufalin selectively inhibits the growth of various lines of human cancer cells (Yeh et al. 2003), especially of leukemic cells (Addya et al. 2004). It can induce leukemic cells,

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such as HL-60, ML1 into apoptosis (Masuda et al. 1995). Electrorotation (ROT) of particles, caused by the interaction between rotating non-uniform alternating current (AC) electrical fields and field-induced polarization of particles, has been applied for the non-invasive manipulation and characterization of individual biological cells, and provides an opportunity for the dielectric characterization of different cell types or cells in different physiological states within a cell mixture. Some of the different cell types, including murine lymphocytes (Hu et al. 1990), yeast (Huang et al. 1992), human erythrocytes (Gimsa et al. 1996), and a number of cancer cell lines (Huang et al. 1996), have been investigated by the ROT method. However, this is the first time that ROT has been used to monitor changes in TCM-treated cell membrane during apoptosis. We constructed a high throughput ROT array chip to overcome the low throughput of the previous ROT devices with a single working unit and examined the changes in cell membrane capacitance and conductance accompanying apoptosis of HL-60 cells induced by bufalin. It can help to quantitatively analyze toxic chemicals effect during identification of novel anti-leukemic compounds from TCM.

Materials and methods

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extracted according to the method of Kundu et al. (2005). Identical amounts of DNA were loaded on a 1.5% agarose gel, containing 3 mg ethidium bromide/ ml, and DNA fragments were separated by electrophoresis. Cell positioning and ROT measurement The biochip-based device is shown in Fig. 1 and our routine ROT procedure (Wu et al. 2005) was used for cell dielectric characterization. For each experiment, 50 ml cell suspension composed of 8.5% (w/v) sucrose + 0.3% (w/v) dextrose with a conductivity of 355 mS/cm was pipetted into the concave edge of the poly(methol methacrylate) (PMMA) cover and was absorbed on to the effective area by capillary action. A negative dielectrophoretic (nDEP) force was used to position the cells within the central region of the electrodes by applying the adjacent electrodes with opposite phase sine waves (DEP Power B, Capitalbio, Beijing, China) (Yu et al. 2004). This could avoid the translational motion of cells caused by the non-uniformity of electric field in the previous ROT measurements (Hu et al. 1990; Huang et al. 1992, 1996; Gimsa et al. 1996). Then, a rotating electric field was established by applying four sine waves in phase quadrature with a frequency range of 100 Hz–25 MHz, at four points per decade. Cell motion was visualized by using microscopy (Leica

Cell culture and bufalin treatment Human promyelocytic leukemia cell line HL-60 (ATCC, Rockville, MD) was cultured according to the methods of Wang et al. (2002). Bufalin stock solutions were prepared in dimethyl sulfoxide (DMSO). When bufalin was added and incubated with cells, in the control experiment (no aspirin present) the cells were exposed to a corresponding dose of DMSO. The incubation times and concentrations of aspirin are shown with the results. MTT assay and cell DNA analysis HL-60 cells were plated at 106 cells/ml in a 96-well plate and measured using the MTT assay method (Yin et al. 2006). The formation of DNA ladders was also examined during the apoptosis progression of HL-60 cells after bufalin treatments. DNA was

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Fig. 1 Electrorotation (ROT) chip and Poly(methol methacrylate) (PMMA) cover. The device was constructed by covering a PMMA cover piece with pre-fabricated microstructures against a glass substrate whose upper surface was plated with a 12 · 2 array of microelectrodes. The electrode array was comprised of plain parallel elements connected alternately to bus lines on either side of a printed circuit board (PCB) (scale bar = 1 cm)

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DMRE, Leica Microsystems, Germany) with a color CCD (Panasonic WV-GP410, Japan). The diameters of cells, calibrated against a micrometer, was determined from its image on the TV monitor; the rotation speed was determined using a stopwatch. The whole measurement was finished within 2 h. Scanning electron microscopy (SEM) Cells from untreated and treated samples were washed in serum-free RPMI and resuspended in 8.75% (w/v) sucrose solution (280 mOs/kg) for 15 min. Cells were fixed in a 3% (v/v) glutaraldehyde in PBS for 1 h, rinsed in PBS and exposed to 1% osmium tetroxide for a further hour. The cells were dehydrated by sequential immersion in 30%, 50%, 70%, 90% and 2· 100% ethanol, followed by a 50/50 ethanol/acetone solution and then 100% acetone followed by sputter coating with gold. Images of cells were taken by using a scanning electron microscope (Fei Quanta 200, Philips Ltd., Netherlands) at a magnification of 8,000 at 10 kV.

Fig. 2 Inhibition effect of bufalin on the growth of HL-60 cells. HL-60 cells were seeded to 96 well plates at a density of 1 · 106 cells/ml and exposed to bufalin or DMSO (0.01%, v/v) for 6 h ( ), 12 h ( ), 24 h ( ), and 48 h ( ). The MTT assay was carried out as described in the Materials and Methods section, and the values are means ± SD of three replicates

Results and discussion Effect of bufalin on the growth of HL-60 and its apoptosis analysis The effect of bufalin on the growth of HL-60 cells was measured using MTT assay at various times and doses, as shown in Fig. 2. When the concentration of bufalin was under 5 nM, the MTT activity increased during the period from 6 to 48 h; i.e., the cells grew. However, when the concentration of bufalin was 10 nM, cell growth was inhibited and they began to die after 24 h. At higher concentrations, bufalin was cytotoxic to cells. Figure 3 shows that 10 nM bufalin can induce HL60 cells apoptosis, as demonstrated by the extensive ladders of DNA fragmentation of the genome. After 6 h treatment, there was a faint DNA ladder, indicating double strand breakage in a significant proportion of cells, and these increased in intensity over 24 h. These data indicated that the apoptosis process was induced at least as early as 6 h and continued progressively with increasing time of exposure.

Fig. 3 DNA fragment formation in HL-60 cells after 10 nM bufalin treatment for 0 h (lane 1), 6 h (lane 2), 9 h (lane 3), 12 h (lane 4) and 24 h (lane 5). Lane 6 was the DNA Marker DL2000 (Takara, Dalian, China). The sizes of relevant marker bands were indicated in base pairs

ROT spectra and data analysis The theory of ROT was given in full detail in previous publications (Wang et al. 1997). In our work, a single shell dielectric model (Huang et al. 1996), in which a cell was considered to be a conducting sphere (cell interior) surrounded by a

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single, thin, dielectric shell (cell membrane), was used to characterize the cell dielectric properties. An optimization algorithm was used to determine the best fit to experimental data and thereby to provide estimates for the dielectric parameters of the cells from the measured ROT spectra. The ROT spectra of untreated HL-60 cells and those treated by bufalin are shown in Fig. 4. We undertook the same experiment four times with consistent results though only one set is shown in the figure. Spectra for the control samples (no bufalin present) incubated for 3, 6, 12, 24, 48 h were almost identical, indicating a reasonably high degree of homogeneity. The shapes of spectral curves of the HL-60 cells were similar to that of some other types of mammalian cells observed previously by other groups (Kurschner et al. 1998). As shown in Fig. 4, the cell rotations occurred in the opposite direction to the applied rotating field (anti-field rotation) below 3 MHz, and in the same direction to the field (co-field rotation) above that frequency at a suspension conductivity of 355 mS/cm. The untreated cells exhibited two relaxation peaks with an anti-field rotation peak and a co-field rotation peak. However, there were distinct differences within the several types of spectra obtained from untreated and apoptotic cells. The anti-field peak of ROT spectra decreased gradually as the time or dose of bufalintreatment increased, and the anti-field peak of ROT spectra shifted progressively towards lower frequencies following the increasing time or dose of the bufalin-treatment. Changes of the dielectric properties for HL-60 cells Membrane capacitance is a measure of the area of cell membrane that acts as a barrier toward, and can accumulate ionic charges in the suspending medium in response to the applied electrical field. On the other hand, due to the near-insulating nature of the membrane lipid bilayer, the conductance mainly reflects the net transport of ionic species across the plasma membrane through the charge carrier transportation system (pores, ion carriers, channels and pumps) under the influence of the applied electrical field (Gascoyne et al. 1997). The corresponding dielectric parameters derived from the best fit curves to the ROT spectra according

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Fig. 4 ROT spectra of HL-60 cells treated by bufalin. (a) The ROT spectra obtained from the cells treated for 0 h (d), 3 h ( ) 6 h ( ), 12 h ( ), 24 h ( ), and 48 h ( ) with 10 nM bufalin. (b) The ROT spectra obtained from the cells treated for 24 h with a series of concentrations of bufalin at 0 (d), 5 nM ( ), 10 nM ( ), 20 nM ( ), and 50 nM ( ). The anti-field peak of ROT spectra decreased gradually as the time or dose of bufalin-treatment increase; meanwhile, the anti-field peak shifted progressively toward lower frequencies as the time or dose of bufalin-treatment increase. The cell samples were suspended and measured in an isotonic sucrose/dextrose medium having an electric conductivity of 355 mS/cm (dot line for experimental data, and real line for theoretical line)

to a single-shell dielectric model (Huang et al. 1996) are summarized in Tables 1 and 2 for the apoptotic HL-60 cells induced by bufalin. These values are close to the data detected by DEP cross frequency measurements (Wang et al. 2002). Figure 5 shows the alterations in the membrane capacitance and conductance over the time and dose course of inducing cell apoptosis.

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untreated cells were covered with abundant microvilli (Fig. 6a). After 12 h treatment with bufalin, the number of microvilli was greatly decreased and bleb formation could be observed (Fig. 6b). This phenomenon was progressively accompanied by an increase in treatment time (Fig. 6c, d). After 48 h, most of the Table 2 The dielectric properties of cell membrane (Cmem) and cell membrane conductance (Gmem) for normal and apoptotic HL-60 cells treated with bufalin in a dose-dependent manner Cmem (mF/m2)

Gmem (·10 6) (mS/m2)

Untreated

15.4 ± 0.8

2.25 ± 1.1

24 h, 5 nM

12 ± 0.8

2.25 ± 1.0

24 h, 10 nM

7 ± 0.7

3.80 ± 1.1

24 h, 20 nM

5.5 ± 0.8

4.50 ± 1.2

24 h, 50 nM

3.4 ± 0.6

8.20 ± 1.3

17

6

15

5

13

4

11 3

9

2

Table 1 The dielectric properties of cell membrane (Cmem) and cell membrane conductance (Gmem) for normal and apoptotic HL-60 cells treated with bufalin at 10 nM in a timedependent manner Time (h)

Cmem (mF/m2)

Gmem (·10 6) (mS/m2)

Untreated

15.6 ± 0.9

2.25 ± 1.1

3

15.0 ± 0.8

2.25 ± 1.1

6

12.8 ± 0.8

2.25 ± 1.0

12

10.4 ± 0.7

2.67 ± 1.1

24

7.5 ± 0.8

3.50 ± 1.2

48

6.4 ± 0.6

4.20 ± 1.3

b 2

Cmem (mF/m )

The morphological study of HL-60 cells by SEM showed the ultra-structural changes were characteristic of apoptosis (see Fig. 6). The untreated cells appeared to be homogeneous in both size (*6 mm diam.) and surface morphology. The surfaces of

0

3

6 12 24 Time of treatment (h)

50

17

12

7

2

0

5 10 20 Dose of treatment (nM)

50

1

10 9 8 7 6 5 4 3 2 1

2

5

Morphologic study of HL-60 cells by scanning electron microscopy (SEM)

6

7

Gmem (*10 S/m )

a

Gmem ( * 1 0 6 m S/m2 )

Time and dose

Cmem (m F/m 2 )

The ROT measurement revealed that the specific membrane capacitance of untreated HL-60 cells was approximately 15.6 ± 0.9 mF/m2 which decreased to 6.4 ± 0.6 mF/m2 after 48 h treatment with 10 nM bufalin (see Fig. 5a). The changes in membrane capacitance were up to about 60% between the initial value and the value after 48 h treatment of bufalin and this is comparable with previous results (Ratanachoo et al. 2002). Tables 1 and 2, and Fig. 5 show that the specific membrane conductance increased in both time- and dose-dependent manners, but these changes occurred later than the observed changes in membrane capacitance. During the first 12 h of treatment, the membrane conductance was (2.25 ± 1.1) · 106 mS/m2 and was little altered compared with the untreated cells but, after 48 h treatment, the membrane conductance reached (4.20 ± 1.3) · 106 mS/m2 (Table 1 and Fig. 5a). This increase might result from injury to the genome and gene transcription system or the membrane barrier function may change because of looser packing of phospholipids and increased field-dependent ion migration through ion channels during apoptosis (Wang et al. 2002). Similar phenomena were also observed during the dose-dependency experiment in our experiments, as shown in Table 2 and Fig. 5(b).

Fig. 5 Changes of the dielectric properties in cell membrane of apoptotic HL-60 cells. (a) The changes of membrane capacitance ( ) and membrane conductance ( ) resulted from bufalin treatments in a time-dependent manner. (b) The changes of membrane capacitance ( ) and membrane conductance ( ) resulted from bufalin treatments in a dose-dependent manner. The cell-specific membrane capacitance decreased as apoptosis progressed, while the membrane conductance increased

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progression of apoptosis these changes in membrane morphology mirrored the measured changes in membrane capacitance (defined as the capacitance per unit area of the membrane) determined by ROT measurements. The dielectric properties of a cell result from the physicochemical properties of the cell which respond sensitively to the changes in cell morphology and physiology. Furthermore, the dielectric alterations of the apoptotic cells can be detected by our ROT method as early as 3 h after bufalin treatment. This indicates that our ROT method is more sensitive than the MTT assay (Fig. 2) and be closely associated in time with the DNA fragmentation, in which a faint DNA ladders appear after 6 h treatment (Fig. 3).

Conclusions

Fig. 6 The SEM of HL-60 cells after treatment with 10 nM bufalin for 0 h (a), 12 h (b), 24 h (c), 48 h (d). Following bufalin treatment, the cells revealed progressive changes in cell membrane morphology as apoptosis developed (scale bar = 5 mm)

cell surface was predominantly smooth with few microvilli (Fig. 6d). The loss of cell surface area accompanying the decreasing morphological complexity was consistent with the observed decrease in membrane capacitance during the progression of bufalin induced apoptosis of HL-60 cells, as shown in Figs. 5 and 6. Previous studies have demonstrated that a shorttime pretreatment of HL-60 cells with bufalin can cause fragmentation of genomic DNA (Masuda et al. 1995), which normally induces apoptosis (Ratanachoo et al. 2002; Wang et al. 2002) and leads to changes in cell morphology, cell surface complexity and membrane properties, such as microvilli, ruffles and folds (Huang et al. 1996; Wang et al. 2002). The SEM results, as shown in Fig. 6, confirmed that a decreases in the density of complex surface morphology of HL-60 cells occurred during the apoptosis progression of HL-60 cells. We believe that over the

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This study demonstrated the capabilities of a high throughput ROT chip, with which we monitored the dynamics of bufalin-treated HL-60 cells. The effect of the various times and doses of bufalin to induce HL-60 cells apoptosis have been investigated using this apparatus. Our results showed that the membrane dielectric properties reflect biophysical alterations in the cell membrane that appear while undergoing apoptosis induced by bufalin. The method offers advantages of speed, sensitivity and scale compared with conventional viability tests, such as MTT assay and DNA fragment formation. The non-invasive performance and the quantitative nature of ROT method, make the device not only may serve for the quantitative analysis of toxic chemicals effects in terms of the kinetics of the development of cell damage but also in the monitoring of cell viability during drug discovery from TCM. Acknowledgements The authors would like to thank Drs. Xiaosheng Guan, Lihua Huang and Guanbin Zhang for helpful discussions; Dr. Keith Mitchelson for his assistance with manuscript presentation. This work was supported by the National Hi-Tech Program (No. 2002AA2Z2011) from the Department of Science and Technology, China.

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