396
Int. J. Nano and Biomaterials, Vol. 2, Nos. 1/2/3/4/5, 2009
FT-IR spectroscopy as a tool for identification of
apoptosis-induced structural changes in A549 cells
treated with PM 701
Gehan Abdel-Raouf Ahmed*
Biochemistry Department, Faculty of Science,
King Abdulaziz University,
P.O Box 42805, Jeddah 21551, Saudi Arabia
E-mail: [email protected]
*Corresponding author
Faten Abdul Rahman Khorshid
Medical Biology Department, Faculty of Medicine,
King Abdulaziz University,
P.O Box 42805, Jeddah 21551, Saudi Arabia
E-mail: [email protected]
Taha Abdullah Kumosani
Head of Biochemistry Department, Faculty of Science,
King Abdulaziz University,
P.O Box 80203, Jeddah 21589, Saudi Arabia
E-mail: [email protected]
Abstract: Fourier transform infrared (FTIR) spectroscopy has been found
useful for detecting apoptotic changes induced in human lung cancer cells
(A549) that treated with PM 701 (natural product) in tissue culture level.
Characteristic bands alterations were identified in the apoptotic cells arising
from cellular protein, lipid and DNA, there were specific changes that affected
the secondary structure of proteins in the apoptotic lung cells appeared in the
FTIR spectra confirmed with the second derivative analysis. To follow the
induced changes in the apoptotic A549 cells, different band absorbance ratios
were calculated for different times up to 24 hours. The results show that there is
a marked decrease in the phosphate absorbance ratio from nucleic acids, the
minimum value was observed at five minutes of PM 701 treatment meanwhile
there is a sharp increase in the lipid-protein ratio the maximum value was
obtained for cells treated at five minutes with PM 701. This study suggests that
FTIR spectroscopy could possibly be used to monitor apoptosis in A549 cells
and prove the efficiency of our new drug PM 701 in cancer treatment.
Keywords: FTIR spectroscopy; apoptosis; A549 cells; structural changes;
quantitative analysis.
Reference to this paper should be made as follows: Raouf, G.A., Khorsid, F.A.
and Kumosani, T.A. (2009) ‘FT-IR spectroscopy as a tool for identification of
apoptosis-induced structural changes in A549 cells treated with PM 701’,
Int. J. Nano and Biomaterials, Vol. 2, Nos. 1/2/3/4/5, pp.396–408.
Copyright © 2009 Inderscience Enterprises Ltd.
FT-IR spectroscopy as a tool for identification
397
Biographical notes: Gehan Abdel-Raouf is an Assistant Professor in the
Medical Physics Department, Faculty of Medicine, King Abdulaziz University,
Jeddah, Saudi Arabia. Her research interests include bio-molecular structure,
drug interaction and application of vibrational spectroscopy in medicine.
Faten Khorshid is an Associate Professor in the Medical Biology Department,
Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.
Taha Kumosani is a Professor and Head of the Biochemistry Department,
Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.
1
Introduction
Apoptosis, as a pre-programmed physiological mode of cell death, plays an important
role in the pathogenesis and progression of cancer. Understanding of the basic
mechanisms that underlie apoptosis will point to potentially new targets of therapeutic
treatment of diseases that show an imbalance between cell proliferation and cell loss. As
an active process, apoptosis involves biochemical changes on three essential cellular
components, DNA, protein and lipid. There are three steps in apoptosis, the initiation
phase triggered by a stimulus received by the cell, the effector or decision phase during
which the cell commits itself to live or to die and the degradation phase when the cells
acquire the morphological and biochemical hallmarks of apoptosis. Common biochemical
events include changes in cell membrane potential, altered levels of protein and RNA
biosynthesis and degradation of DNA into small fragments. DNA fragmentation,
activation of caspases and externalisation of phosphoatidylserine are considered as three
hallmarks of apoptosis (Willingham, 1999; Coelho et al., 2000). Morphologically, cells
undergoing apoptosis display membrane blebbing, cell shrinkage, nuclear condensation,
fragmentation and packaging of cellular material into apoptotic bodies. Clearly, it would
be desirable to detect apoptosis at an early stage, i.e., before phase that entails the visible
changes of apoptosis.
Consequently, a method that can detect all three processes simultaneously would
provide a useful addition for the study of apoptosis. Because chemotherapeutic agents
exert their in vivo anti-tumour activity by triggering apoptosis in susceptible
tumour cells, the in vitro measurement of drug-induced apoptosis should provide a
mechanism-based test for the chemosensitivity of the tumour cells isolated from an
individual patient and may represent a prognostic marker for the treatment response
(Kravtsov et al., 1998; Hannun, 1997). Furthermore, it was demonstrated that IR
spectroscopy can distinguish the different phases of cell division when examining
purified populations (Boydston-White et al., 1999) and render diagnoses and even
prognoses on the outcome of disease (Schulz et al., 1997). The understanding of infrared
spectra of individual cells becomes of paramount importance for the interpretation of
spectra of tissue samples. Cancer becomes one of the leading cause of death in many
countries over the world. Early diagnosis and treatment increases the chances of survival
and full recovery (Dukor, 2001). Fourier transform infrared (FTIR) spectroscopy has
been used by chemists as a powerful tool to characterise inorganic and organic
compounds (Khorsid and Mushref, 2006). It has been applied in biology for studying the
398
G.A. Raouf et al.
structure and conformation of molecules like proteins (Cooper and Knutson, 1995),
nucleic acids (Mantsch and Chapman, 1996) and lipids (Brandenburg and Seydel, 1998).
Herein, we continue the work done earlier by Khorsid and Mushref (2006) to explore
the potential of PM 701 as anti-cancer agent, which produce apoptosis in human lung
cancer cells (A549) dry samples at the tissue culture level together with the potential of
infrared (FTIR) spectroscopy as a means for the early detection of apoptosis.
2
Materials and methods
•
Media: The following commercially available media were prepared according to
published literature, these include: ordinary media, minimal essential medium
(MEM) (10% FCS); MEM is a rich, multipurpose medium that was used for
cultivation of human lung cancer cells (A549). Phosphate buffer saline (PBS) is a
phosphate-buffered physiological saline solution. Calcium and magnesium free
solution trypsin.
•
Examined media: PM 701 is a natural product, easily available, cheap, sterile and
non-toxic according to our chemical and microbiological testing. PM 701 added to
the ordinary media with ratio 1:100.
•
Human lung cancer cells line: A549 cells, non-small cell carcinoma was obtained
from cell strain from American Type Cultural Collection (ATCC), available in the
cell bank of Tissue Culture Unit, King Fahd Medical Research Center (KAU,
Jeddah).
•
In vitro proliferation of cells
1
2
3
•
A549 was suspended in culture medium MEM
the cells were dispensed in 3X (six wells plate), 1 × 105/ml in each well
each group of cells was incubated 24 hours in suitable media and apoptosis was
induced by incubating with dissolved lyophilised PM 701 at different time
points.
Infrared spectroscopy and statistical analysis: At various time points, the cells
treated with PM 701 were washed twice (by centrifugation for three minutes at
300g). The cell pellet was kept at –20°C before freeze drying, then the dried A549
cells dispersed in potassium bromide (KBr) to form 1% concentration discs. The
FTIR spectra from three searate samples were recorded in absorbance form using
Shimadzu FTIR-8400s spectrophotometer. For each examined sample, the spectrum
was taken as the average of three different measurements. The spectra were obtained
in the wave number range of 400 cm–1 to 4,000 cm–1 with an average of 20 scans to
incease the signal to noise ratio and at spectral resolution of 4 cm–1. All the samples
were baseline corrected and normailsed by using IR solution software before any
measurements. The averages of the normalised spectra were used for subsequent
analysis. Other normalisation method such as amide I for the 800 cm–1 to 1,800 cm–1
region have also been tested and gave negligible changes in the results. To extract
the maximum information from the IR bands, we applied Fourier deconvolution to
all spectra before using difference methods between the spectra under investigations.
Second derivative spectra were tacking in comparison studies. The parameters
FT-IR spectroscopy as a tool for identification
399
studied were nucleic acid ratio, lipid-protein ratio (Mourant et al., 2003; Gaingneaux
et al., 2002) and phosphate levels (Ramesh et al., 2003; Gasparri and Muzio, 2003).
3
Results and discussions
Representative IR spectra of A549 cells before and five minutes to 30 minutes and 24
hours after treatment with PM 701 are shown in Figure 1 (samples treated for 15 minute
and 2 hours are not shown for clarity reasons). It is observed from the figure that there are
dynamic changes in the intensities of the normalised spectra during treatment at various
wave numbers. Since the absorbance is dependent on the concentration of various
metabolites, alteration of any/all of the metabolites with respect to each other would
manifest themselves as changes in the spectra. These several important changes in the IR
spectra are related to molecular vibrations of proteins, lipids and DNA. Thus, drug affect
A549 cells, cause biochemical changes in A549 cells and induces apoptosis (Khorshid
and Mushref, 2006).
Figure 1
Representative IR spectra of A549 cells before and after treatment with PM701 for
different times (see online version for colours)
control
5min.
30min.
24hr
1
Abs
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1800
1700
1600
1500
1400
1300
1200
1100
1000
900
1/cm
The amide I band (which provides information about the overall protein secondary
structure in the cells (Rigas and Wong, 1992; Surewicz and Mantsch, 1988; Liu and
Mantsch, 2001) arising from the amide C = O stretching vibration of the peptide groups
in all proteins (Figure 1) shifts from at 1,652 cm–1 in the control cells to 1,645 cm–1 in the
cells treated with PM 701 for five minutes and 15 minutes. These amide I frequencies are
compatible with the fact that overall protein structure in the control cells consists
primarily of α-helix, whereas at the first step of apoptosis apoptotic A549 cells have a
relatively high proportion of β-sheet after five minutes of treatment while they have a
relatively high proportion of unordered proteins constituents after 24 hours of PM 701
400
G.A. Raouf et al.
treatment. Since apoptosis involves the modification of existing proteins, as well as the
synthesis of new proteins (Liu et al., 2001), we analysed the changes of protein structure
upon PM 701 treatment in more detail. Figure 2 shows the mean spectra of control and
PM 701 treated cells in the region of the amide I (1,600 cm–1 to 1,700 cm–1) and amide II
(1,500 cm–1 to 1,600 cm–1) bands. Spectra are shown as second derivative, a method
commonly used for narrowing broad IR bands for better visualisation. It is obvious from
the figure that the changes in the secondary structure of the proteins are time dependant.
The corresponding absorbance’s bands of amide I and amide II at the examined time of
treatment are presented in Table 1. A similar shift in the amide I and amide II bands
during apoptosis was reported earlier (Liu et al., 2001).
Figure 2
Mean spectra for control and PM 701-treated A549 cells for different times up to 24
hours as second derivatives (see online version for colours)
Abs/(1/cm)^2
5min.2nd deriv.
cont.2nd derivative
15min. 2nd deriv.
30min. Second Derivative
24hr. Second Derivative
0.0006
0.0005
0.0004
0.0003
0.0002
0.0001
8.13152E-20
-0.0001
-0.0002
-0.0003
-0.0004
-0.0005
-0.0006
-0.0007
-0.0008
-0.0009
1750 1700 1650 1600 1550 1500 1450 1400 1350 1300
1/cm
Table 1
The different absorbance peaks of the amide I and amide II bands as appeared in the
second derivatives spectra
Time of PM 701
treatment
Control
Amide I absorbance band (cm–1)
1,652s
1.684 sh
Amide II absorbance band (cm–1)
1,542s
1,516m
5 min
1,636s
1,656s
1,681 sh
1,542s
1,519 sh
1,572 w,.sh
15 min
1,636s
1,656s
1,681 s.sh
1,542s
1,522 sh
1,572 s.sh
30 min
1,653s
1,685 sh
1,542
1,515
2 hrs
1,650
1,681 s.sh
1,544
1,513
24 hrs
1,660s
1,636 sh
1,544
1,524
1,681 sh
Note: s = strong; w =weak; m = medium; sh = shoulder.
1,570 sh
FT-IR spectroscopy as a tool for identification
401
The effect of apoptosis on DNA can be identified from IR bands associated with
vibrations of various structural groups in DNA such as the band at 972 cm–1 (marked as
C-C/C-O), a single bond stretching vibration characteristic of the deoxyribose suger
moiety in DNA. The bands at 1,087 cm–1 and 1,238 cm–1 (marked as sPO2– and as PO2–),
due to vibrations of the phosphodiester groups in DNA. The FTIR spectra in Figure 1
clearly showed decrease in the absorbance of phosphate bands corresponding to nucleic
acids. Also, the band at 967 cm–1 accounting for symmetric stretching vibration of the
phosphodiester bonds in nucleic acids showed reduction in the intensity after PM 170
treatment and disappeared up to 24 hours of drug treatment.
The absorbance ratios RIII and RIV calculated as the ratio of absorbance at
1,238 cm–1 over the absorbance at 2,925 cm–1 and the ratio of absorbance at 1,087 cm–1
over the absorbance at 2,925 cm–1 respectively are plotted versus the time of treatment in
Figure 3. The figure shows that the behaviour of the absorbance ratios RIII and RIV are
similarly with all time of treatment. There are dramatic decreases in the values of these
ratios at five minutes of treatment. There are observed increase over the minimum value
in these ratios in late time of treatment.
Absorbance Ratios (RIII,RIV)
Figure 3
The changes in RIII (A1238 cm–1/A2925 cm–1), RIV (A1087 cm–1/A2925 cm–1) ratios
with PM 701 treating time (see online version for colours)
RIII
RIV
0.66
0.64
0.62
0.6
0.58
0.56
0.54
0.52
cont.
5min
15min
30min
2hr
24hr
time of treatment
Note: The values are average of three different measurements on each sample.
The absorbance ratio R (amide II/amide I) calculated as the ratio of absorbance at
1,542 cm–1 over the absorbance at 1,652 cm–1 and plotted versus the time of treatment is
shown in Figure 4. This ratio has been indicated to represent the variation in nucleic acids
(Benedetti et al., 1997). The figure shows a marked decrease in the value of this ratio for
the samples treated during the first 15 minutes than that of the control sample. The
minimum value was achieved after five minutes of treatment followed by an increase in
the value of this ratio at 30 minutes of treatment. The amide II/amide I ratio was
dramatically decreases up to 24 hours of treatment.
402
Figure 4
G.A. Raouf et al.
The changes in amide II/amide I (A1,542 cm–1/A1,652 cm–1) ratio with PM 701 treating
time (see online version for colours)
amide II/amide (A1542cm1/A1652cm-1) ratio
0.75
0.7
0.65
0.6
0.55
0.5
cont.
5min
15min
30min
2hr
24hr
time of treatment
Note: The values are average of three different measurements on each sample.
Figure 5
IR difference spectrum between control and PM 701-treated A549 cells for 24 hours
(see online version for colours)
0.3
Abs
0.275
0.25
0.225
0.2
0.175
0.15
0.125
0.1
0.075
0.05
0.025
6.93889E-18
-0.025
-0.05
-0.075
1750
1650
1550
1450
1350
1250
1150
1050
950
Subtraction of contro l Deco nvo lutio n from 5min Deconvolutio 1/cm
(a)
FT-IR spectroscopy as a tool for identification
Figure 5
403
IR difference spectrum between control and PM 701-treated A549 cells for 24 hrs
(continued) (see online version for colours)
0.3
Abs
0.25
0.2
0.15
0.1
0.05
1.38778E-17
-0.05
-0.1
-0.15
1875
1725
1575
1425
Subtraction of cont. from 24hr.
1275
1125
975 900
1/cm
(b)
Figures 5(a) and 5(b) illustrates the difference spectrum between treated samples for
five minutes, 24 hours and the control A549 cells, generated by subtracting the mean
spectrum of control cells from that of PM 701 treated A549 cells. From the difference
spectrum, one can clearly observe that DNA content in the cells treated with PM 701 for
24 hours has significantly decreased. These results are in good agreement with the results
obtained earlier by Khorsid and Mushref (2006) who studied the effect of PM 701 as
anti-cancer agent in vitro on the same type of cancer A549 cells. Live images of the cells
showed that the severe lethal effects of PM701 on cancer cells started immediately after
five minutes to six minutes since adding the examined substrate. They continued that the
cancer cells incubated in PM 701 showed that the substrate attacks the cell’s nuclei,
which is indicated by the appearance of pale ring around the nucleus of the lung cancer
cell after 30 minutes of incubation. This leads to the degradation of the cells, which could
not be reversed to recover the cells by re-growing the cells in ordinary media again.
These results are also consistent with the finding that at the early stage of apoptosis,
initiation phase is triggered by a stimulus received by the cell, this can be explained by
the first five minutes to 15 minutes of PM 701 treatment where the R ratio is decreasing
together with the observed band shift in the amide I and amide II (Liu and Mantsch,
2001; Liu et al., 2001). These results are also in good consistence with the results
obtained by the electron microscope (EM)
404
Figure 6
G.A. Raouf et al.
EM showed the morphological hallmarks features that characterise apoptosis, as shown
by chromatin condensation and membrane blebbing in treated cells with PM 701
(arrows) data not bubleshed (see online version for colours)
(Figure 6) showed the morphological hallmarks features that characterise apoptosis, as
shown by chromatin condensation and membrane blebbing in treated cells with PM 701
(arrows) (data not published). The sharp decrease in the value of (I Amide II/I Amide I)
ratio at five minutes and 15 minutes of treatment and the marked decrease in RIII and
RIV values at the same time of treatment may be due to condensation of chromatin
during the early stage of apoptosis, which in turn decreases the path length. This is may
referred to dark DNA, one would expect that less photons are transmitted within the
strong absorption bands of DNA (which make up the chromatin) by particles of such
relatively high optical density. Furthermore, given the number of transitions and the
width of the DNA and protein bands, the entire highly condensed chromatin would be
virtually opaque (Brian et al., 2005; Jamin et al., 2003). The relatively increase in the RIII
and RIV values than the minimum value when the sample treated up to 24 hours with PM
701 may referred to the degradation of DNA. (Jamin et al., 2003) reported a large
FT-IR spectroscopy as a tool for identification
405
increase in the DNA signals of apoptotic cells. In apoptosis, DNA is degraded into
oligonucleotides with a few hundreds of base pairs that subsequently diffuse out of the
nucleus. This observation suggests that the DNA, although highly condensed in the
nucleus, has a low absorption, yet its degradation products in late apoptosis are detected
by IR spectroscopy. The decrease in protein concentration was evident with lower
intensity for the amide II band.
The lipids in the plasma membrane are composed mainly of phospholipids that
determine membrane stability, fluidity and membrane enzymatic activity. Thus,
monitoring lipid absorbance in the treated samples is an important for detecting
apoptosis. Lipid changes in the treated cells can be derived from other IR bands as (figure
not shown) the dominant lipid bands in the range 2,800 cm–1 to 3,000cm–1 originate from
CH stretching vibrations of the fatty acyl chains of all cellular lipids (unsaturated lipids
show = CH band at 3,012 cm–1, while the band at 1,740 cm–1 originates from lipid ester
C = O groups (appeared in the mean spectra as a weak shoulder and as a strong band in
the second derivative spectra. The treatment of A549 cells with PM 701 increases the
CH2 content, this is indicated by the marked increase in lipid protein ratio calculated as
the ratio of absorbance at 2,854 cm–1 over the absorbance at 1,400 cm–1 CH2/CH3 and
illustrated in Figure 7. It is obvious that (all) lipid bands show a tendency to increase in
intensity, starting five minutes after the treatment. We also followed the overall cellular
lipid changes after PM 701 treatment. The treatment of A549 cells with PM 701 increases
the lipid content as most of these lipid bands show positive intensities in the difference
spectrum (not shown). On the other hand, the characteristic CH3 group stretching
vibrations at 2,870 cm–1 and 2,954 cm–1 show negative band intensities in the difference
spectrum which may reflect structural changes of phospholipids rather than relative
changes of lipid in the treated cells (Liu and Mantsch, 2001; Liu et al., 2001). It is noted
that the initial value of lipid-protein ratio 2,854 cm–1/1,400cm–1 is greatly lower than the
final value. This may be due to membrane blebbing and increases content of CH2. The
PM 701 contains proteins (paper in progress), which may contact with A549 cell
membrane and may form some sort of an ion channel or pore in the cell membrane
thereby, enhancing the ion permeability of the membrane and destroying the cell, i.e.,
induce apoptosis (Matsuzaki, 1998; Shai, 1999). Zasloff (1987) suggested that the peptide
could perturb membrane functions responsible for osmotic balance. The relative observed
decrease after five minutes of treatment in (Figure 8) may be due to that pore formation is
a transient process observable mainly during the early stage of the peptide-membrane
interactions (Matsuzaki, 1998). Matsuzaki (1998) proved that the peptides translocate
into the inner monolayers after forming the pores. There was a temporal relationship
between the onset of apoptosis induced by PM 701 treatment and a 1.4 fold increase in
the lipid-protein ratio (CH2/CH3). The increase in the methylene 1H NMR signal intensity
could be attributable to changes in plasma membrane fluidity or could also be because of
an increase in the amount of fatty acids within the lipid bilayer. Francis et al. (1996)
cleared that the dominant methylene 1H NMR resonance shown in their studies do not
appear without a large fraction of apoptotic cells. They suggested that the increase in the
methylene in the lipid-protein ratio is associated with the onset of apoptosis and
independent of the stimuli used to induce apoptotic cell death.
406
G.A. Raouf et al.
Figure 7
Variation in lipid-protein ratio (A2,854 cm–1/1,400 cm–1) with the treating time (see
online version for colours)
Lipid/protein(A2854cm-1/A1400cm-1)
1.2
1.15
1.1
1.05
1
0.95
0.9
0.85
cont.
5min
15min
30min
time of treatment
2hr
24hr
Notes: The values are average of three different measurements on each sample. The solid
line gives the trend.
The dying cell (apoptotic cell) shows two characteristic spectral signatures indicative of
death (Jamin et al., 2003). First, the centroids of the protein amide I and II peaks shift to
lower frequency, indicating a change in the overall protein conformational states within
the cells. Second, the appearance of a peak at around 1,743 cm–1.These observation can
now be used as signatures of cell death (Hoi-Ying et al., 2000). Moreover, Liu et al.
(1997) found that FTIR spectroscopy could be used for the detection of drug
sensitive/resistant leukemic cells.
4
Conclusions
To conclude, we can state that PM 701 can induce apoptosis to A549 cells and FTIR
spectroscopy is able to identify overall changes in the apoptotic cells in the form of dry
samples. The dominant protein secondary structure shifts from α-helix structure to
unordered, indicating an alterated protein profile in the apoptotic cells. In addition, the
total cellular lipid content increases starting from the first five minutes up to 24 hours of
treatment while the amount of DNA decreases dramatically during treatment with PM70
as observed in difference spectrum. It should mentioned here that condensation of
chromatins in early stage of apoptosis was detected in the first time by IR spectroscopy
for dry samples after five minutes of PM 701 treatment and confirmed by the previously
published paper (Khorshid and Mushref, 2006).
FT-IR spectroscopy as a tool for identification
407
Acknowledgements
The authors would like to thank Prof. Mansour Soliman, Head of the Clinical
Pharmacology for his cooperation, Dr. Sawsan Jalalah for carrying on EM study,
Dr. Ahmed Shaker for the pharmacological advices and formulation and
Ms. Hanady Quashaqury from Tissue Culture Unit for carrying on the tissue culture
technique. This research work was supported by Scientific Research Council from King
Abdulaziz University grant no. N02/427 and King Abdulaziz City for Science and
Technology grant no. M S 11-23.
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