In the present study, we examined the1H NMR ML signals of cultured tumour cells, specifically HeLa and Keywords cell cycle; cell metabolism; lipids; magnetic resonance spectroscopy; tumou
Trang 1in cells from human adenocarcinomas
Anna Maria Luciani1, Sveva Grande1, Alessandra Palma1, Antonella Rosi1, Claudio Giovannini2, Orazio Sapora3, Vincenza Viti1and Laura Guidoni1
1 Dipartimento di Tecnologie e Salute and INFN Gruppo Collegato Sanita`, Istituto Superiore di Sanita`, Rome, Italy
2 Dipartimento di Sanita` Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanita`, Rome, Italy
3 Dipartimento di Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanita`, Rome, Italy
Among the different molecules showing intense and
narrow peaks in the 1H magnetic resonance spectra,
much attention has been devoted to the fatty acid
sig-nals from mobile lipids (MLs), which are characterized
by high mobility and, thus, differently from most cell
lipids, are visible in high resolution magnetic resonance
spectra High-intensity MLs are often observed in
pro-liferating cells and in tumour cells [1–4] Many studies
have found that the onset of apoptosis is accompanied
by an increase in ML intensity [5–7], although other
studies have not [8,9]
A number of studies have been performed in view of
the possible use of MLs as spectroscopic markers of
cell fate, although a clear explanation of their behav-iour has not yet been provided Essentially, two differ-ent localizations have been proposed In mammalian cells, ML resonances arise from lipids that are either present as microdomains with high mobility embedded
in the plasma membrane bilayer [9] or exist in cytosolic lipid droplets, mostly consisting of triglycerides (TGs) [10,11] Some studies have found that the concentra-tion of total cell TGs was consistent with the intensity
of ML signals [11], whereas, more recently, changes in the size of lipid droplets were suggested [12]
In the present study, we examined the1H NMR ML signals of cultured tumour cells, specifically HeLa and
Keywords
cell cycle; cell metabolism; lipids; magnetic
resonance spectroscopy; tumour cell lines
Correspondence
L Guidoni, Dipartimento di Tecnologie e
Salute, Istituto Superiore di Sanita`, 00161
Rome, Italy
Fax: +39 06 4938 7075
Tel: +39 06 4990 2804
E-mail: guidoni@iss.it
(Received 5 November 2008, revised 15
December 2008, accepted 22 December
2008)
doi:10.1111/j.1742-4658.2009.06869.x
Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti-cancer treatments Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death The present study explored the rela-tionship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation A sub-G1 peak was present only
in the radiosensitive HeLa cells Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation-induced growth arrest The intensities
of triglyceride signals in the spectra from extracted total lipids changed concurrently The increase in lipid peak intensities did not correlate with the apoptotic fate Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids This is observed for the first time in cells that are different from adipocytes
Abbreviations
GPC, glycerophosphorylcholine; IR, intensity ratio; ML, mobile lipid; PC, phosporylcholine; PCA, perchloric acid; PL, phospholipids;
TG, triglycerides.
Trang 2MCF-7 cells from human cancers In a previous study
[13], we demonstrated that these cells display intensity
modulation of ML signals as a periodic event
accom-panying cell growth In a recent study [14], we also
showed that these cells are differently prone to
apopto-sis induced by treatment with gamma rays We
associ-ated the radiation-induced apoptosis of tumour cells
with the level of reduced glutathione, as detected by
1H NMR [14]
These cells were therefore considered for use in
mag-netic resonance spectroscopy analysis for the detection
of possible different trends in ML spectral features
during cell growth in culture and after
radiation-induced arrest in proliferation Within this framework,
a similar modulation of ML signals with growth was
observed in both cell lines, whereas radiation-induced
arrest in proliferation capacity resulted in a different
pattern Effects on cell cycle frequency were also
observed ML signal intensity modulation was
tenta-tively related to modulation of lipid metabolism
Results
Cell spectra were first examined for signal
quantifica-tion after spectral assignment Subsequently, changes
in signal modulation were monitored when cell growth
was arrested by means of treatment with ionizing radi-ation Finally, a model to fit signal intensity modula-tion was proposed
Analysis of1H NMR spectra – spectral assignments and quantification Both cell lines displayed very similar spectral features Besides other signals, the characteristic peaks from MLs were observed to be in agreement with the data available in the literature for cancer cells [1–4] Under the present experimental conditions, the intense ML signals can be attributed to the fatty acid chains of neutral lipids, mostly TGs, in agreement with the data available in the literature [1,15] and on the basis of our previous observations showing that, in these cells,
TG peaks are more intense in the lipid extract spectra derived from cells with high ML signals compared to spectra with low ML signals [13] This point will be discussed further below
Figure 1 shows an example of the1H NMR spectra from MCF-7 cells at day 3 (Fig 1A) and at day 6 (Fig 1A¢) after seeding It is worth noting that the choline-based signals at 3.2 p.p.m display the same intensity with very different ML signals (Fig 1A,A¢) When the ML signals were intense in the 1D spectra
(p.p.m.)
(p.p.m.) (p.p.m.)
M1 + ML1
A
B
Fig 1 Spectra of MCF-7 cells in different proliferative conditions: 1D 1 H NMR spectra from cells at day 3 after seeding (A) and at day 6 after seeding (A¢) ML signals are at 0.89 p.p.m (ML1), 1.28 p.p.m (ML2) and 1.55 p.p.m (ML3); M1 and M2 from macromolecules are also labelled 2D1H NMR COSY spectra of the same cell samples at day 3 after seeding (B) and at day 6 after seeding (B¢) Cross peak A is from terminal methyl and bulk methylene coupling in ML The reference cross peak from Lys is also labelled Label T refers to the glycerol cross peak of triglycerides The insert shows the glycerol cross peak region of the geminal protons of triglycerides.
Trang 3(Fig 1A), the corresponding 2D COSY spectra were
also characterized by prominent cross peaks from fatty
acid chains (Fig 1B), in agreement with the data
avail-able in the literature [15] and previous observations
[13] The cross peak at 4.07–4.24 p.p.m., generated by
protons in the glycerol backbone of TG, was also
visi-ble only in ML rich spectra (T) Details with respect to
this signal are provided in Fig 1B (insert), which
shows the characteristic cross peak from the geminal
protons of carbons 1 and 3 of glycerol in TG [1]
Cross peaks of lipids, including the T signal, were
absent in cells characterized by low ML signals in 1D
spectra (Fig 1B¢) Very similar behaviour was
observed in HeLa cells according to previously
reported data [4,13]
Signal assignments in cell spectra were performed
after comparison with the spectra from lipid and
per-chloric acid (PCA) extracts, derived from cell samples
grown and harvested under similar conditions, and
with compound spectra Assignments from the
litera-ture were also taken into account [1–4,16] Figure 2
shows typical spectra (1D and 2D COSY) from lipid
and PCA extracts both relative to a MCF-7 cell
sample with high MLs It is worth noting that, in the 2D COSY spectra from extracted lipids, the glycerol geminal cross peaks of 1,3-glycerol protons of TG and
of 1-glycerol protons for phospholipids (PL) are clearly separated, in agreement with the data available in the literature [1]
For peak assignments and intensity quantification, deconvolution of 1D spectra and integration of 2D cross peaks was performed as described in the Experi-mental procedures The signal intensity refers to the peak area in the 1D spectra and to the cross peak inte-gral in the 2D spectra Internal reference signals were used Our study compared samples where cell volumes and cell packing change, which hinders the use of an external reference
The signal at 0.96 p.p.m., present in cells (peak M2
in Fig 1) and PCA extract spectra, derives from poly-peptide chains and was used as the intensity reference for 1D spectra because it is indicative of cell mass As far as the intensity reference for 2D spectra is con-cerned, the sum of peaks of Lys at 1.70–3.00 p.p.m and Ala at 1.48–3.77 p.p.m (Fig 1B) was chosen as the area reference in the 2D COSY spectra
(p.p.m.)
(p.p.m.) (p.p.m.)
(p.p.m.)
A
A′
B
B′
Fig 2 1D 1 H NMR spectra of extracted lipids (A) and PCA extracts (A¢) from one representative sample of MCF-7 cells; 2D 1 H NMR spectra
of extracted lipids (B) and PCA extracts (B¢) from one representative sample of MCF-7 cells The insert shows the glycerol cross peak region
of the geminal protons of TGs and PLs.
Trang 4For 1D spectra, deconvolution of spectra of the type
shown in Fig 1A¢, with ML signals of a very low
intensity, was performed as a first step Deconvolution
of spectra of the type shown in Fig 1A, with intense
ML signals, was then performed starting from the lines
and parameters previously found, by adding the signals
from MLs A typical deconvolution pattern with the
used resonances is shown in Fig 3A The
correspond-ing parameters are given in Table 1 Experiments
con-ducted on different samples derived from the same
culture (at least three samples) demonstrated that the
SD from this procedure did not exceed 0.01 p.p.m for
chemical shifts and 10% for linewidths and intensity
ratios (IRs) On the other hand, the variability of
signal intensities, especially for MLs, exceeded the
measurement error in spectra from samples derived
from different culture, even when cells were harvested under similar growth conditions For this reason, the spectral behaviour over time was compared among samples obtained by cells harvested at different days from the same seeding
Cholesterol peaks were present in lipid extracts at 0.70 and 1.03 p.p.m in the 1D spectra (Fig 2A) and with the typical cross peak at 0.87–1.50 p.p.m in the 2D spectra (Fig 2B) In spectra from cell samples, we could therefore assign the peak at 0.71 p.p.m (Fig 3A and Table 1) to the methyl group in C18 of choles-terol This signal was present in all cell spectra, although it was broader and more intense when ML signals were present, and its IR changed from 0.07 to 0.36 as obtained by 1D spectra deconvolution In par-allel, peaks at 1.03 and 1.50 p.p.m., as obtained from 1D spectra deconvolution (Fig 3A and Table 1), increased, and the cross peak at 0.87–1.50 p.p.m appeared in the cell spectra This latter feature is evident in Fig 1A¢,B¢ The peak at 1.03 p.p.m could therefore be attributed to the methyl group in C19 and
(p.p.m.)
(p.p.m.)
A
B
Fig 3 Example of the deconvolution pattern of the methylene
region in the 1D 1 H NMR spectra of MCF-7 cells (A); integration
regions for selected cross peaks in the 2D 1 H NMR spectra of
MCF-7 cells (B) (Lys1, lysine; A, bulk methylene and terminal
methyl group in fatty acids; E, C4 methylene and C3 methylene in
fatty acids; Lino, linolenic acid) Other ML related cross peaks are
visible as peaks B and F; for assignments, see [2] Rectangles
indi-cate the area used for signal integration.
Table 1 Mean values of parameters d (p.p.m.), Dm (Hz) and IR, after deconvolution (1D) and integration (2D COSY) of spectra, such
as those presented in Fig 3 Values were obtained from the spec-tra of three different samples derived from the same culture The standard deviation was 0.01 p.p.m for chemical shift values and 10% for linewidths and IR Chemical shifts are referring to lactate methyl; IR are calculated with respect to M2 in 1D and to Lys1 + Ala in the 2D spectra.
Trang 5the peak at 1.50 p.p.m to the other bulk protons of
cholesterol
As far as MLs are concerned, spectra with intense
ML signals could be fitted by adding a peak at
1.28 p.p.m., a second peak at 1.31 p.p.m and a peak
at 1.58 p.p.m (Table 1 and Fig 3A) to the spectral
fitting of samples without ML signals The presence of
these peaks was paralleled by the existence of lipid
cross peaks in the 2D spectra (Table 1 and Fig 3B)
The cross peak A, due to the interaction of terminal
methyl group peak at 0.89 p.p.m and the proximal
methylene at 1.28 p.p.m., was used to quantify MLs
because it is representative of the corresponding bulk
fatty acids chains This excludes the contribution from
x-3 fatty acids, where methyl protons at 0.98 p.p.m
are coupled to the allylic methylene at 2.09 p.p.m [16]
The signal at 0.88 p.p.m is mostly from the terminal
CH3 of ML chains, with a minor contribution from
the cholesterol C25 and C26 methyl groups, and from
an unidentified macromolecule methyl group (M1)
This signal is present: (a) in PCA extracts (Fig 2A¢)
and (B) in cells when ML signals are absent
(Fig 1A,B) The same considerations hold for the
peaks at 1.22 and 1.40 p.p.m (Table 1 and Figs 2 and
3) These signals most likely arise from the aggregation
of large molecules because they are characterized by
large linewidths (Table 1 and Figs 2 and 3) Work is in
progress to clarify the nature of these structures
Signal intensities were also measured in the 2D
spec-tra A typical 2D COSY spectrum is shown in Fig 3b,
including the details of the cross peaks examined The
peaks chosen for evaluation are framed with rectangles
that denote the areas used for volume integration
When very intense ML signals are present in the
spec-tra, the signals related to unsaturated fatty acids also
are evident, and are more intense in MCF-7 than in
HeLa cell samples Besides the cross peaks resulting
from the connectivity of the vinyl protons (at
5.35 p.p.m.) to the allylic protons (at 2.05 p.p.m.) in
monounsaturated chains and to the bis-allylic protons
(at 2.80 p.p.m.) in polyunsaturated fatty acids (not
shown), the cross peaks at 1.64–2.09 p.p.m and 1.68–
2.24 p.p.m., attributed to arachidonic acid chains, and
at 0.93–2.04 p.p.m., attributed to linolenic acid chains
on the basis of a comparison with lipid extracts and
from the data available in the literature, are also
clearly visible in Fig 1B,B¢
Experiments on different samples derived from the
same culture (at least three samples) were also
exam-ined to assess measurement errors in the 2D cross peak
integration Under these conditions, errors on cross
peak volumes did not exceed 10%, whereas the
vari-ability of integral values exceeded this error in the
spectra from samples derived from different cultures, even when cells were harvested under similar growth conditions For this reason, the behaviour over time of 2D COSY cell spectra were compared in samples obtained by cells harvested at different days from the same seeding
In the following, ML quantification derives from intensity measurements of the peak at 1.28 p.p.m in 1D spectra and from the integral of cross peak A at 0.89–1.28 p.p.m Cross peaks from PL glycerols (Fig 2B, insert) were never observed in the 2D COSY spectra of cells, even in the presence of very high ML signals in the 1D spectra Cross peaks from glycerol protons of TG in cells were not routinely used for TG quantification because the intensities were much smal-ler and the errors were larger ML signals were then used to monitor TG levels in cells
Biological changes and ML signal modulation with growth and in growth-arrested cells Changes in ML signals were monitored in parallel with cell growth and after cell cycle arrest due to irradiation
Cell proliferation and cell cycle Cell growth behaviour was examined in HeLa and MCF-7 cells Cells were routinely grown as described
in the Experimental procedures Cells were sampled at different days after seeding for both NMR experiments and cell cycle measurements Under the chosen experi-mental conditions, cells were kept in the exponential growth phase (up to 3 days from seeding) Cells were then irradiated with a single dose of 20 Gy (gamma irradiation) to provide growth arrest and cell death with different characteristics in the two cell lines, according to previous observations [14] Figure 4 shows the cell counts as a function of time for one representative experiment in HeLa and MCF-7 cells Compared to control samples, the differences in cell counts were larger in HeLa than in MCF-7 cells Similar behaviour was observed in at least three independent experiments
To assess cellular transcriptional responses to radia-tion-induced DNA damage, we examined cell cycle arrest in MCF-7 and HeLa cells at 1, 2 and 3 days after treatment Both cell lines underwent cycle arrest upon irradiation, with different characteristics Figure 4A¢,B¢ shows the percentage of cell phases of both cell lines observed after 2 days after irradiation at
20 Gy Although both cells were blocked in G2⁄ M, HeLa cells displayed a remarkable decrease in the
Trang 6G1 phase, whereas MCF-7 cells showed G1 block and
a decreased percentage in the S phase compared to
control samples Irradiated HeLa cells showed an
intense sub-G1 peak (> 20%), indicating DNA
frag-mentation and the occurrence of significant apoptosis
This observation is in agreement with previous data
obtained in the same cells by monitoring apoptosis as
the externalization of phosphatidyserine [14]
1H NMR ML signals in intact cells and TG signals in
lipid extracts
To clarify whether intensity changes of ML signals
were mainly related to changes in TG concentration,
to differences in chain mobility due to structural
changes, or to the different size of droplets, as recently
suggested by Quintero et al [12], we compared the
behaviour of the spectra of cells and the total lipid
extracted from cells Irradiation was then used to
arrest cell growth, thus providing a modification of
ML signal intensity in cells Lipids were extracted from
cell samples under identical conditions
In previous experiments on these cell lines [4], we
observed an intensity modulation of ML with cell
growth Consequently, the extent of the variation in
intensity with irradiation was monitored after different
time intervals after irradiation Figure 5A,B shows the
ML signal intensities of 1D spectra from the two
cell lines run at different days Samples of cells grown
under the same conditions were irradiated and the two sets of spectra compared (control and treated samples) Similar data were obtained for 2D measurements (Fig 5A¢,B¢), where the data are from one representa-tive experiment Error bars indicate measurement errors By comparing the results of at least seven inde-pendent experiments, the differences between control and irradiated samples were significant for MCF-7 cells at all time intervals examined On the other hand, statistical significance for HeLa samples was observed at days 2 and 3 after irradiation Figure 5C shows these differences for samples examined 2 days after irradiation
Figure 6 shows the glycerol region from two repre-sentative 1D spectra of total lipids extracted from control and irradiated HeLa (Fig 6A,A¢) and MCF-7 (Fig 6B,B¢) cells A significant change in relative inten-sity of TGs (glycerol proton centred at 4.32 p.p.m.) versus PLs (glycerol proton centred at 4.42 p.p.m.) was found in irradiated samples compared to controls Particularly, TG signals were depressed in HeLa cells (Fig 6A,A¢), whereas an increase was evident in MCF-7 cells (Fig 6B,B¢) Deconvolution of 1D spectra was performed to provide the relative intensities of TG versus PL, which were calculated on sn-1 and sn-3 glycerol signals centred at 4.32 p.p.m for TG and sn-1 glycerol signals at 4.42 p.p.m for PL (Fig 6C) In this experiment, the calculated relative concentration
of TG versus TG + PL was 15% and 12% in MCF-7
A
A′
B
B′
Fig 4 Number of HeLa (A) and MCF-7 (B) cells (N) as a function of time after irradiation for both control (h) and irradiated ( ) samples The solid black line is the fit with an exponential function Percentage of MCF-7 (A¢) and HeLa (B¢) cells in the different cell cycle phases, measured 2 days after irradiation One representative experiment is reported for both control and irradiated samples.
Trang 7and HeLa cells, becoming 20% and 8%, respectively,
after irradiation The standard deviation in repeated
calculations was 2% The relative concentrations of
TG calculated by1H NMR were found to be in
agree-ment with that reported in previous studies [17]
Intensity measurements of 1D signals of sn-2
glycerol protons of TG at 5.29 p.p.m and PL at
5.25 p.p.m., clearly resolved only at 700 MHz,
gave similar results (not shown) This behaviour is
similar to that observed for ML signals in whole
cells (Fig 5)
Total extracted TG may be more abundant with
respect to NMR visible TG in cells For this reason,
the spectra from extracted lipids were not used to
calculate NMR visible TG in intact cells
Lipid metabolites from PCA extracts
To provide further information on lipid metabolism,
the PCA extracts were also analyzed The region of
choline metabolites around 3.2 p.p.m is shown in Fig 7 for PCA extracts of HeLa Mean values of parameters d (p.p.m.), Dm (Hz) and IR were obtained from deconvolution of the 1D spectra (three different samples derived from the same culture) of PCA extracts and are reported in Table 2 The standard deviation was 0.005 p.p.m for chemical shift values and 10% for linewidths and IR
Signals from the headgroups of glycerophosphoryl-choline (GPC) at 3.24 p.p.m., phosporylglycerophosphoryl-choline (PC)
at 3.23 p.p.m and choline at 3.21 p.p.m showed dif-ferent behaviour in the two cell lines after irradiation
In particular, more relevant changes in GPC⁄ PC ratios were observed in irradiated MCF-7 cells (Fig 7B,B¢) with respect to HeLa cells (Fig 7A,A¢), indicating the different equilibrium of catabolism versus anabolism Table 3 reports on the intensity of changes of the choline-based metabolites for the two cell lines after irradiation resulting from fittings of at least three different spectra
A/(Lys + Ala) c A/(Lys + Ala) i
C
Fig 5 Intensity modulation of ML signals
from a representative experiment (1D and
2D 1 H NMR data) for HeLa (A, A¢) and
MCF-7 cells (B, B¢) Spectra were acquired
at different days from seeding for both
control (h) and irradiated ( ) samples
(D = 20 Gy) Errors obtained from spectral
fitting (1D) and integration (2D COSY) are
contained within the symbols (C) Relative
IRs ML ⁄ M (control: white; irradiated: black)
and A ⁄ (Lys + Ala) (controls: dotted white;
irradiated: dotted black) as obtained from 1D
and 2D COSY spectra of HeLa and MCF-7
cell samples Data are the mean ± SD
values of seven independent experiments.
Spectra were acquired on day 5 after
seed-ing and 2 days after irradiation with a sseed-ingle
dose of 20 Gy *P < 0.05 (t-test).
Trang 8Model for the ML signals
To find a possible explanation for the experimental
data, a model for ML intensity modulation is
pro-posed
As previously reported [4,13] growth of MCF-7 and
HeLa cells slows down as cells approach confluence
This finding is in agreement with the observed increase
of the G1 phase in the final days in culture (Fig 4
A¢,B¢) It is reasonable to assume that cell metabolism,
including PL synthesis, slows down accordingly On
the other hand, intensities of ML (in cells) signals,
mainly due to TG according to a previous study [13]
as well as the present study (compare inserts in Figs 1
and 2), are characterized by a nonlinear behaviour
over time in culture (Fig 5) A mechanism that is
more complicated than a simple decrease of lipid
production over time must be therefore envisaged
There is a growing body of evidence indicating that lipid metabolism possesses an articulated role with respect to maintaining cell equilibrium, which takes into consideration both PL synthesis⁄ breakdown and
TG metabolism [18–20] We may infer that there are two mechanisms inside the cell: one relative to the pro-duction of ML (and TG) with a rate constant Rpand one relative to the consumption of ML (and TG) with
a rate constant Rc The signal that we observe in the NMR spectra is due to the net accumulation of reserve lipids and its rate, dML⁄ dt, is given by the difference
of the rate of lipid production Rpand the rate of lipid consumption Rc:
We may assume that both rates Rp and Rc are not constant, but decrease over time in culture as a conse-quence of cell proliferation slowing down A linear dependence of both rates can be assumed:
where p1 and c1 are the production and consumption rates at time = 0, respectively, and p2 and c2 are the changes of production and consumption rates over time
By integrating Eqn (1), we obtain a second degree polynomial function for the lipid accumulation:
MLðtÞ ¼m1t2þm2tþm3 ð4Þ
where m1= (c2) p2)⁄ 2, m2= (p1) c1) and therefore are related to the equilibrium between lipid production and consumption, and m3is the starting ML value The best fit with Eqn (4) of data ML⁄ M and
A⁄ (Lys + Ala) versus time from independent experi-ments on HeLa and MCF-7 cells gave a chi-square value that was always < 1 for both the 1D and 2D COSY experiments Figure 5A,A¢,B,B¢ shows these fittings for a representative experiment for both cell lines Figure 8 reports the parameters m1, m2 and m3 (mean ± SD) as obtained from fittings with Eqn (4) for the data obtained from ten samples for HeLa and ten samples for MCF-7 of cells harvested irrespective
of the growth phase
(p.p.m.) (p.p.m.)
(p.p.m.)
C
Fig 6 Glycerol region of representative 1D1H NMR spectra from
total lipids extracted from HeLa (A, A¢) and MCF-7 cells (B, B¢).
Lower traces: control samples (A, B), upper traces: irradiated
(D = 20 Gy) samples (A¢, B¢) Spectra were acquired 2 days after
irradiation Example deconvolution pattern (C) of 1D 1 H NMR
spectrum for sn-2 glycerol protons of TGs (couple of doublets
centered at 4.32 p.p.m., coupling 12 Hz and 4 Hz) and PLs (couple
of doublets centered at 4.42 p.p.m., coupling 12 and 3 Hz)
Accord-ing to this deconvolution pattern, in this spectrum, the ratio
TG ⁄ (PL + TG) was 0.34.
Trang 9The minimum value tmof the parabolic fitting curve,
representing the time value for which production and
consumption rates are equal, and the corresponding
lipid value MLmare also reported
1D and 2D data were in good agreement, although
the starting intensity values and values at the minimum
were different, reflecting different pools of ML inside
the cells By changing the seeding density, the minimum
of the curve was shifted, but the shape of the curves did not change It is worth noting that m1 was always positive (i.e c2was always greater than p2), whereas m2 was always negative (i.e c1was always greater than p1) For m1> 0, the parabola was concave upward For
m2< 0, the minimum was after time zero
(p.p.m.) (p.p.m.)
Fig 7 1D 1 H NMR spectra of the
choline-related metabolites region from PCA
extracts of HeLa (A, A¢) and MCF-7 cells
(B, B¢) Lower traces: control samples
(A, B), upper traces (A¢, B¢): irradiated
samples (D = 20 Gy) Spectra were acquired
2 days after irradiation Deconvolution
pattern of the same region (C) and of the
reference signal (C¢).
Table 2 Mean values of parameters d (p.p.m.), Dm (Hz) and IR
after deconvolution of 1D spectra from PCA extracts of HeLa cells
(Fig 7C,C¢) Values were obtained from the spectra of three
differ-ent samples derived from the same culture The standard deviation
was 0.005 p.p.m for chemical shift values and 10% for linewidths
and IR.
Table 3 Mean values (three independent experiments) of IRs of the choline-related metabolites from PCA extracts of HeLa and MCF-7 cells for control and irradiated samples The standard devia-tion was 10%.
HeLacells
MCF-7 cells
Trang 10Although the t-test showed that differences were not
significant between the two groups (HeLa and
MCF-7), m1 values were generally higher in MCF-7 than in
HeLa, thus reflecting the tendency for higher final
values of ML signal intensities in MCF-7 cells (Fig 8)
Cells were then irradiated to arrest cell growth; data
from irradiated cells could be still fitted through Eqn
(4), as shown in Fig 5 Figure 9 shows the parameters
(mean ± SD) obtained by fitting the data from five
independent experiments on HeLa cells and five
inde-pendent experiments on MCF-7 cells (both irradiated
and non-irradiated cells) The parameter m1 decreased
and m2 increased after irradiation in both cell lines
(Fig 9), but only the m2 increase in MCF-7 cells was
statistically significant in independent experiments
(P < 0.05; t-test) This may be due either to an
increase in c1 (consumption rate at time = 0) or a
decrease in p1 (production rate at time = 0) The
relevant m2 increase in MCF-7 cells produced the
great increase of ML signals with respect to controls
(Fig 5B,B¢) In some experiments conducted on
MCF-7 cells, the minimum of the parabolic curve at
time > 0 was no more evident and m2 became
posi-tive Finally, tmshifted to lower values in MCF-7 cells
and the MLm value was considerably higher in
irradiated MCF-7 cells compared to controls
Discussion
The appearance of intense signals from bulk methylene
of fatty acid chains in high resolution 1H NMR spec-tra of cells has been studied subsequent to the first observations being made in cancer cells, lymphocytes and developing cells On the other hand, a correlation
of the intensity of these signals with metabolic parame-ters is not straightforward Some studies noted that the signal intensity of bulk methylene is influenced by cell proliferation, as T lymphocyte activation [21] and
in tumour cells by the different proliferation state [4,22]; other studies found that the onset of apoptosis correlates with the increase of lipid signals, whereas others did not [5–9] Finally, some studies found that these signals can be affected by extreme pH conditions, which is more likely due to the effects of low pH on cell proliferation [22]
In the present study, ionizing radiation was used to affect cell growth and induce cell death in cells show-ing different attitudes with respect to undergoshow-ing radi-ation-induced apoptosis The relevant sub-G1 peak observed in the cell cycle profile of radiation-arrested HeLa cells (Fig 4A¢) points to significant apoptosis, in agreement with the previously observed radiation-induced apoptosis determined by phosphatidylserine
5.0
0.0
–5.0
–10.0
5.0
0.0
–5.0
Fig 8 Parameters m 1 , m 2 and m 3 obtained from fitting with Eqn (4) ML data, as in Fig 5 The minimum values (tm) of the para-bolic fitting curve and the corresponding lipid values (MLm) are also reported Bars represent the mean ± SD of ten indepen-dent experiments for each cell line.
5
0
–5
–10
5 0 –5 5 0 –5
5
0
–5
–10
Fig 9 Parameters m1, m2and m3obtained from fitting with Eqn (4) ML data, as in Fig 5, for irradiated (I) and non-irradiated (C) HeLa and MCF-7 cells The minimum values (t m ) of the parabolic fitting curve and the corresponding lipid values (MLm) are also reported Bars represent the mean ± SD of five independent experiments on each cell line *P < 0.05 (t-test).