1. Art and Diseño

Effect of temperature on surface properties of cervical tissue
homogenate and organic phase monolayers
A. Preetha a , R. Banerjee a,∗ , N. Huilgol b
a
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai 400076, India
b Division of Radiation Oncology, Nanavati Hospital, Mumbai, India
Abstract
The temperature dependence of Langmuir monolayers of normal and cancerous human cervical tissues and their organic phases between
temperatures of 37 and 45 ◦ C was evaluated. Analysis of the surface pressure–area isotherms revealed significantly different increase in fluidity of
the cancerous cervical tissue monolayer at 42 ◦ C as opposed to the normal cervical tissue monolayers (p < 0.05). Similarly, in the case of cervical
cancerous organic phase monolayers significant increase of fluidity was observed at 40 ◦ C whereas no such change was observed in the normal
cervical organic phase monolayers. The effect of temperature was found to be different in cancerous and normal cervical tissues and this may be
due to the different lipid profiles in them. Cancerous cervical tissues had 1.8-fold higher total lipids as compared to the normals. Similarly, the PC,
PE, PI, PG, SM and PS levels in cancerous cervical tissues were 3.6, 2.0, 2.3, 4.7, 1.7 and 2.2 times higher than those of normal cervical tissues,
respectively. Significant cancer–normal difference in minimum surface tension and hysteresis area was found at all temperatures studied for both
tissue homogenates and organic phases. For example, cancerous tissue homogenates showed minimum surface tensions of 51.9 ± 4.6, 54.4 ± 5.9,
57.6 ± 6.0 and 51.9 ± 5.6 mN/m at temperatures 37, 40, 42 and 45 ◦ C whereas the corresponding values for normal cervical tissue homogenates
were 39.3 ± 3.6, 39.2 ± 3.7, 39.2 ± 3.8 and 39.1 ± 3.6, respectively. The fluidity change at hyperthermic range of temperature can be correlated to
the increased efficiency of drug on combination therapy with hyperthermia. These results may have implications in manipulating the fluidity of
cervical cancer tissue membranes for better permeability thereby leading to better therapeutic strategies for cervical cancer.
Keywords: Tissue tensiometry; Surface pressure–area isotherm; Temperature; Hysteresis; Hyperthermia; Cervical cancer
1. Introduction
Cervical cancer is one among the leading cause of cancer mortality in women [1]. Presently multimodal treatment of cancer
is gaining attention and hyperthermia is being widely explored
as an adjunct to radiotherapy and chemotherapy. A review by
Hildebrandt et al. [2] reported that several clinical trials have
demonstrated improved survival rates for pelvic tumor patients
treated with combined radiotherapy and hyperthermia. A phase
II study reported an improved outcome of a triple modality
treatment, involving cisplatin, radiotherapy and hyperthermia,
in patients having advanced cervical cancer and a phase III
trial of the same for cervical cancer is on [3]. These literature
evidences suggest the role of hyperthermia in cervical cancer
treatment. Hyperthermia is the treatment of malignant diseases
by the application of heat. Clinically relevant hyperthermic temperature range is between 40 and 43 ◦ C [2,4]. Hyperthermia itself
has been shown to be cytotoxic and several chemotherapeutic
agents in combination with hyperthermia have shown additive
cytotoxic effects [5–7].
Temperature has the ability to perturb biological membranes. For example, non-lethal doses of temperature produced
a remodeling of the composition and alkyl chain unsaturation of
membrane lipids in E. coli which manifested as membrane fluidization and permeabilization [8]. Monolayers at an air–liquid
interface are convenient models for understanding the behavior
of many natural self-organized systems like biological membranes because they allow simulation of biological conditions
[9]. Hence Langmuir monolayers are selected as model systems
for monitoring the surface properties of cancerous and normal
cervical membranes with respect to the change of temperature
in this study.
13
Kazakov et al. [10] showed that the dynamic surface tension
(with respect to time) of serum from cervical cancer patients was
lower as compared to that of normals. During radiotherapy, the
surface tension was found to shift towards that of normals. Thus,
it was shown that interfacial tensiometry of human biological
fluids provided data with the potential to differentiate various
pathologies and to monitor the treatment processes. Recently,
the surface tension of astrocytoma tissue aggregates was found
to be related to the invasive potential of the cancers [11]. Studies
by Foty et al. [12] have established that dexamethasone treatment of HT-1080 human fibrosarcoma cells caused a 2.5-fold
higher cell cohesivity or surface tension which was correlated
with a reduced invasiveness of the tumors. Surface tension measurements of lung carcinoma cell aggregates also demonstrated
the role of cadherins in suppression of invasion [13]. However,
these studies do not use Langmuir monolayers for evaluation of
surface properties. Our earlier studies using Langmuir monolayers at 37 ◦ C, established significantly lower surface activity of
cancerous cervical tissue homogenate monolayers as compared
to the normal cervical tissue homogenate monolayers. Similarly,
the surface activity of normal cervical organic phase was found
to be significantly lower as compared to the cancerous cervical
organic phase [14]. This paper deals with the effect of temperature on the surface properties of the normal as well as cancerous
cervical tissue homogenate and their organic phase monolayers. The temperatures evaluated were 37, 40, 42 and 45 ◦ C as
they are the clinically relevant temperatures for the hyperthermia
treatment of cancer.
2. Experimental details
2.1. Collection, preparation and extraction of tissue
Human biopsy specimens of cervical cancerous tissues
(n = 15) and normal cervical tissues (n = 15) were obtained from
the Radiation Oncology Division of Nanavati Hospital, Mumbai, India. The use of human tissue biopsies was approved by the
ethical committee of the hospital. All cancerous samples were
collected prior to any treatment. All cases were of stage III squamous cell carcinoma of cervix. Normal controls were obtained
from hysterectomy patients having non-cervical disorders. All
the normal cases were reported to be free from malignancy by
the histopathology analysis and the cervical portion was normal. Equal weights and equal number of cells of cancerous and
normal tissues were homogenized for evaluation.
All the tissue samples were washed thoroughly with normal
saline, dried on a tissue paper and weighed. The weighed samples were processed by using liquid nitrogen and dissolved in
measured volumes of normal saline to get a tissue homogenate
of known concentration. The tissue homogenate was kept at
−10 ◦ C till experimentation and all the experiments were performed within 10 days of sample collection. It was confirmed
that the surface activity of fresh and stored tissue homogenate
was not altered within 10 days. The organic phases containing
the lipophilic components and the aqueous phases containing
the lipophobic parts of the tissue homogenates were separated
by using Bligh–Dyer extraction procedure [15]. Briefly, 0.8 ml
of the tissue homogenate was treated with chloroform and
methanol in the ratio 1: 2 (v/v) and agitated well to get a single phase. To this 1: 1 (v/v) chloroform: water was added and
agitated well for 5 min followed by centrifugation at 100 × g
for 10 min, to achieve good phase separation. The separated
organic phases were stored at −10 ◦ C till experimentation. All
measurements were conducted within 24 h of extraction.
2.2. Laboratory chemicals
HPLC grade methanol and chloroform (for tissue extraction
purpose), HPLC grade potassium hydroxide and ammonium
molybdate were purchased from Loba chime, Mumbai, India.
AR grade methanol and acetone for cleaning of Langmuir
trough and potassium dihydrogenphosphate for phosphorus
assay standard were purchased from SRL, Mumbai, India. Phosphatidylcholine for testing the accuracy of phosphorus assay and
cholesterol for cholesterol assay standards were obtained from
Sigma-Aldrich Co. (St. Louis, USA). High purity water purified
by a Milli Q Plus water purifier system (Milli pore, USA), with
a resistivity of 18.2 M cm, was used for all experiments.
2.3. Lipid quantification
The quantification of total lipid, total cholesterol, total phospholipid and individual phospholipids in cancerous and normal
cervical tissues was also performed. Gravimetric method was
used for the determination of the amount of total lipids extracted
from cervical tissues. The total cholesterol content was quantified by a colorimetric assay using ortho-phthalaldehyde in
glacial acetic acid and concentrated sulphuric acid. The total
phospholipid content was quantified by malachite green phosphorus assay [14]. The separation of individual phospholipids
from the tissue extracts was done by modified touchstone’s
thin layer chromatographic (TLC) method and the individual
phospholipid quantification was done by phosphorus assay after
separation by TLC [16].
2.4. Monolayer experiments
Monolayer studies were performed by using a computer
controlled LB film balance (KSV Mini trough model, KSV
Instruments, Finland). The trough is placed on an antivibration table, which is enclosed by an environmental chamber.
The Teflon coated trough is equipped with two delrin barriers (for monolayer compression and expansion) and the entire
trough is surrounded by a water jacket, providing temperature control. A Wilhelmy plate balance with a platinum plate
(19.62 mm × 10 mm) is used for sensing the surface pressure.
Before each monolayer experiment, the trough and barriers
were thoroughly cleaned by organic solvents (methanol and acetone) and deionized water in sequence several times. Highly pure
deionized water, having resistivity 18.2 M cm, was the subphase in all experiments. The surface pressure–area isotherms
were recorded at temperatures 37, 40, 42 and 45 ◦ C. The temperature of the subphase was maintained at the desired value with
the help of an external circulating water bath and the variation of
14
the temperature during the course of experiment was ±0.5 ◦ C.
The surface was cleaned with the help of an aspirator and a zero
reading of the surface pressure ensured the cleanliness. One milligram of tissue homogenate/organic phase (100 ␮l of 10 mg/ml
solution) was spread as tiny droplets on the surface of the subphase using a Hamilton syringe. In the case of organic phase,
30 min wait time was given for the evaporation of the organic
solvents. The surface pressure–area isotherms were recorded by
continuous compression and expansion of the monolayer for
three cycles (1 cycle = 1 compression + 1 expansion) with a barrier speed of 120 mm/min. The maximum relative area change
during compression was 86.5% and the surface pressure was
measured with a sensitivity of 0.004 mN/m.
Table 1
Lipid profiles of cancerous and normal human cervical tissues
Lipids
Represented as mg/g of tissue
Cancerous
Total lipid
Total phospholipid
Cholesterol
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylinositol
Phosphatidylglycerol
Phosphatidylserine
Sphingomyelin
60.55
4.83
23.02
1.44
0.40
0.82
0.61
0.48
1.03
±
±
±
±
±
±
±
±
±
0.04*
0.08*
0.28*
0.03*
0.03*
0.06*
0.02*
0.01*
0.02*
Normal
33.15
1.94
15.45
0.40
0.20
0.36
0.13
0.22
0.61
±
±
±
±
±
±
±
±
±
0.02
0.04
0.09
0.01
0.01
0.02
0.02
0.02
0.01
Values expressed as mean ± standard deviations.
* Represents significantly different values from the normal controls (p < 0.05).
2.5. Calculation of parameters
From the surface pressure–area isotherms obtained, the
following parameters were calculated. The minimum surface
tension (γ min ) was calculated as γ min = γ s − πmax where πmax is
the maximum surface pressures and γ s is the surface tension of
the sub phase. The hysteresis area ( G) is the difference between
the free energy of compression and free energy of expansion
which are calculated from the area under the corresponding surface pressure–area isotherms. The effect of temperature on γ min
and G of the monolayers was evaluated in this study.
2.6. Statistical analysis
Fifteen human cervical cancerous tissues and 15 normal
human cervical tissues were characterized in this study and
for each monolayer; tensiometric parameters were calculated
as explained. The data are expressed as mean ± standard deviation. The effect of temperature on cancerous and normal tissue
homogenates and their organic phases was compared by Student’s t-test at 95% confidence level. A paired t-test was used as
the tensiometric profile of same tissue was evaluated at different
temperatures ranging from 37 to 45 ◦ C. A statistical comparison
of the surface activity of cancerous and normal cervical tissues
at each of the temperatures studied was performed by unpaired
Student’s t-test at 95% confidence level.
Fig. 1 depicts the surface pressure–area isotherms of cancerous and normal human cervical tissue homogenate monolayers
at different temperatures. In Fig. 1, all the isotherms are means
of 15 tissue homogenate data. The reproducibility of the surface
pressure–area isotherms was checked by repeated recordings
and the relative standard deviation in surface pressure was
found to be ≤2%. The shape of the isotherm was not significantly affected by the increase of temperature in both cancerous
and normal tissue homogenates. The normal tissue homogenate
isotherms showed an increase in surface pressure from 90% area
of compression at all temperatures studied. On the other hand,
the cancerous tissue homogenate isotherms showed horizontal
regions till 55% area of compression at all temperatures studied.
Characterization of the organic phase monolayers of cancerous and normal cervical tissues was also done at different
temperatures. The mean surface pressure–area isotherms of
organic phases of cancerous and normal cervical tissues at various temperatures are shown in Fig. 2. The mean compression
isotherm of the cancerous organic phase showed an increase in
surface pressure at the beginning of compression but the normal
organic phase isotherms showed a similar rise below 70% area
that is on further compression.
3. Results
The lipid levels of cancerous and normal cervical tissues
are summarized in Table 1. One can find 1.8-fold higher total
lipids in cancerous cervical tissues as compared to the normals.
Similarly, the total phospholipid and total cholesterol content
of the cancerous cervical tissue was 2.5- and 1.5-fold higher
than that of the normal tissue, respectively. All the individual phospholipids quantified in this study were found to be
higher in cancerous cervical tissues compared to normal tissues.
The phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylserine (PS) and sphingomyelin (SM) levels in cancerous
cervical tissues were 3.6, 2.0, 2.3, 4.7, 2.2 and 1.7 times higher
than those of normal cervical tissues, respectively.
Fig. 1. Surface pressure–area isotherms of cervical tissue homogenates at different temperatures. All the isotherms are means of 15 tissue homogenate data
from first compression isotherms.
15
Fig. 2. Surface pressure–area isotherms of cervical tissue organic phases at
different temperatures. All the isotherms are means of 15 organic phase data
from first compression isotherms.
Fig. 3 shows the effect of temperature on minimum surface
tension of cancerous and normal cervical tissue homogenates.
The normalized minimum surface tensions with respect to the
value at 37 ◦ C have been plotted against the temperature. For all
cancerous tissue homogenates the normalized minimum surface
tension shows a maximum value at 42 ◦ C. Significant elevation
in minimum surface tension of cancerous tissue homogenates
was observed on rise of temperature from 37 to 42 ◦ C whereas
no significant change in minimum surface tension was found in
normal tissue homogenate at these temperatures as evidenced
by t-test at 95% confidence level.
The effect of temperature on minimum surface tension
of organic phases of cancerous and normal cervical tissues
has been depicted in Fig. 4. The minimum surface tension
achieved at 40 ◦ C was significantly higher that that at 37 ◦ C
for all the 15 cancerous organic phase samples. In case
of normal cervical organic phase, no significant change in
minimum surface tension was observed on rise of temperature from 37 to 45 ◦ C as indicated by the near unity values
of normalized minimum surface tension at all temperatures
studied.
The bar diagram in Fig. 5 compares the minimum surface
tension values of tissue homogenates and organic phases in cancerous and normal cervical tissues at various temperatures. As
can be seen from Fig. 5, the minimum surface tension of cancerous tissue homogenate was significantly higher than that of the
normal tissue homogenate at all temperatures studied (p < 0.05).
In case of organic phases, the minimum surface tension of the
cancerous organic phase was significantly lower than that of the
normal cervical organic phase at all temperatures studied.
Table 2 depicts the effect of temperature on hysteresis area
of cancerous and normal tissue homogenates and their organic
phases. In cancerous and normal tissue homogenate monolayers, the hysteresis area was not significantly affected by a rise in
temperature from 37 to 45 ◦ C. Similar result was also observed
in case of the cancerous and normal organic phase monolayers.
However, the hysteresis area of the cancerous tissue homogenate
monolayers was significantly lower than that of the normal ones
at all temperatures studied. In case of organic phases, the hysteresis area of cancerous organic phase was significantly higher than
that of the normals at all the temperatures studied as evidenced
by t-test (p < 0.05).
4. Discussion
Plateaus were observed in the surface pressure–area
isotherms of cancerous tissue homogenates and normal organic
phases above 55 and 70% areas, respectively, at all temperatures studied (Figs. 1 and 2). These plateaus denote the gaseous
to liquid expanded phase transitions in these monolayers. These
plateaus were unaltered at all the temperatures studied. In normal
tissue homogenates and cancerous organic phases, an increase
in surface pressure was observed from ∼90% area of compression indicating molecular ordering on film compression. This
Fig. 3. Effect of temperature on minimum surface tension of cancerous and normal cervical tissue homogenate monolayers. Each point represents a surface
pressure–area isotherm recorded at that specified temperature and each curve represents a tissue sample.
16
Fig. 4. Effect of temperature on minimum surface tension of cancerous and normal cervical organic phase monolayers. Each point represents a surface pressure–area
isotherm recorded at that specified temperature and each curve represents an organic phase from a tissue sample.
Fig. 5. Comparison of minimum surface tension of tissue homogenates and organic phases in cancerous and normal tissues at various temperatures. Values represented
as mean ± standard deviation of 15 sample data.
was unaffected by the rise of temperature within the range of
37–45 ◦ C.
Minimum surface tension represents the maximum possible monolayer packing. The higher the γ min , more fluid
will be the monolayer. The γ min values of cancerous and
normal tissue homogenates indicate that the normal tissue
homogenate monolayer is more fluid than the cancerous tissue homogenate. However, the opposite is true in case of the
cancerous and normal organic phase monolayers. Significant
differences in γ min values indicate the change in molecular
ordering and molecular interactions of the monolayer components [17,18].
From Fig. 3 it can be observed that the minimum surface
tension was significantly higher at 42 ◦ C than that at 37 ◦ C for
the cancerous tissue homogenates. This suggests a fluidizing
effect of temperature in cervical cancerous tissue homogenate
monolayers at 42 ◦ C. No such effect was observed in the normal
cervical tissue homogenate monolayers. The cancerous organic
phase monolayers had a higher minimum surface tension at
40 ◦ C compared to that at 37 ◦ C. This suggests a fluidizing effect
Table 2
Effect of temperature on hysteresis area of cervical monolayers
Monolayers
37 ◦ C
Cancerous tissue homogenate
Normal tissue homogenate
Cancerous organic phase
Normal organic phase
16.7
90.3
78.9
31.4
±
±
±
±
40 ◦ C
7.8*
17.7
9.6*
6.8
16.9
88.9
86.3
39.8
±
±
±
±
42 ◦ C
7.8*
14.5
10.2*
7.9
Values expressed as mean ± standard deviations.
* Represents significantly different values from the normal controls at the same temperature (p < 0.05).
20.1
91.2
79.8
40.6
±
±
±
±
45 ◦ C
9.0*
11.6
11.6*
10.6
18.4
87.0
81.3
38.9
±
±
±
±
9.2*
10.1
16.7*
9.6
17
in cancerous organic phase at 40 ◦ C. No change in minimum
surface tension was observed in the normal organic phases at
all the temperatures studied. The significantly different surface
activities of cancerous and normal tissues between 37 and 45 ◦ C
suggest a fluidizing effect at 42 ◦ C in cervical cancerous tissue
homogenates which may be of relevance in hyperthermia. An
increase in fluidity of the cell membrane due to hyperthermia
has also been reported in the literature [2].
The effect of temperature on cancerous and normal cervical monolayers was found to be different. This may be due to
the composition differences of the cancerous and normal monolayers. The lipid profiles in the cancerous and normal cervical
tissue was found to vary as described in Table 1. Membrane lipids
are reported to play a remarkable role in thermosensitivity and
thermotolerance of mammalian cells [19]. The lipid profiles in
normal and cancerous cervical tissues may influence the effect of
temperature on the tissue homogenate and organic phase monolayers. The acyl chain composition was found to significantly
affect the temperature dependence of Langmuir monolayers of
sphingomyelin between 10 and 30 ◦ C [20]. If a change in acyl
chain of a lipid could produce remarkable difference in thermotropic behavior of the lipid monolayer, then the difference in
lipid profile may produce different surface properties for mixed
monolayers, like the ones in the present study, with respect to
temperature.
Cervical cancerous tissue homogenates had lower surface
pressure and hence a higher minimum surface tension than
that of normal tissue homogenates at all temperatures studied (Fig. 5). This suggests that cervical cancerous tissue
homogenates formed more fluid monolayers than their normal
counter parts. Similarly, membranes of lung cancerous tissues
have been reported to be more fluid than the corresponding normal tissues and high plasma membrane fluidity of lung tumors
has been associated with a poor prognosis [21,22]. However, in
case of organic phases this trend is reversed and the higher surface activity or lower minimum surface tension of the organic
phase of cervical cancerous tissue as compared to normal at all
temperatures studied, may be explained by the higher amounts
of surface active lipids in the cancerous organic phase. The
organic phase of cervical cancerous tissues was more surface
active than that of the normals whereas the normal cervical tissue homogenate was more surface active than the cancerous
tissue homogenate. This result suggests that not only the surface
active lipids but also interaction between the lipid and non-lipid
components of the tissue determines its surface activity.
Reproducible hysteresis area across compression–expansion
cycles represents the slow respreading of the molecules on
expansion. A higher hysteresis area is indicative of a condensed
phase of the monolayer [23]. In this study hysteresis area was
reproducible across cycles. An increase of temperature from 37
to 45 ◦ C did not significantly change the hysteresis area in any
of the monolayers studied. This indicates that the stability of
the condensed phase of the monolayers was not significantly
affected by temperature rise from 37 to 45 ◦ C. However, the
hysteresis area of cancerous tissue homogenate was significantly
lower compared to that of the normal tissue homogenates, indicating the lower stability of the cancerous tissue homogenate
monolayers as compared to the normal controls. The hysteresis
area values also revealed the higher stability of the cancerous
organic phase monolayers as compared to normal organic phase
monolayers.
5. Summary
The minimum surface tension of cancerous tissue
homogenates was significantly higher at 42 ◦ C than at 37 ◦ C.
No such increase in minimum surface tension was observed in
normal tissue homogenates between temperatures of 37 and
45 ◦ C. This increased surface tension due to higher temperature
may be of relevance in hyperthermia. In case of organic phases,
the minimum surface tension of cancerous organic phase was
significantly higher at 40 ◦ C than that at 37 ◦ C. No such effect
was observed in normal organic phase monolayers between
temperatures of 37 and 45 ◦ C. The increase in minimum surface
tension observed in cancerous tissue homogenates and organic
phases at temperatures of 42 and 40 ◦ C, respectively, could be
indicative of an increased fluidity at these temperatures and may
be correlated to the increased efficacy of chemotherapy when
combined with hyperthermia. Such changes in surface tension
and fluidity at 40–42 ◦ C may have relevance to the response of
the tissues to hyperthermia. In short, the results of this study
may have implications in manipulating the fluidity of cervical
cancerous membranes to improve the efficacy of hyperthermia
based therapeutic strategies in cervical cancer.
Acknowledgments
We thank Dr. Priti D. Galvankar and Dr. Ketaki R. Shah of
Nanavati Hospital, Mumbai, India for their help in collection
and pathological analysis of cervical tissues.
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