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UHM 2004, Vol. 31, No. 2 – HBO2 on human oral cancer cells
The effect of hyperbaric oxygen on human oral
cancer cells.
T.B. SUN1, R.L. CHEN2, Y.H. HSU3
1
Division of Plastic Surgery and Center for Hyperbaric Oxygen Therapy; 2Center for Bone Marrow Transplantation;
Department of Pathology, Tzu Chi Medical Center and Institute of Medical Sciences, Tzu Chi University, Hualien,
Taiwan.
3
Sun T.B., Chen R.L., Hsu, Y.H., The effect of hyperbaric oxygen on human oral cancer cells. Undersea
Hyperb Med 2004, 31(2):255-264. Discoveries of the beneficial cellular and biochemical effects have
strengthened the rationale for the administration of hyperbaric oxygen therapy (HBO2) as an adjunctive
therapy for the treatment of osteoradionecrosis (ORN). Malignancies, however, are considered a
contraindication for HBO2 because of the possible tumor-promoting effects. The aim of this study was to
examine the effects of HBO2 therapy on tumor weight, and to measure the progression of apoptosis and
tumor cell proliferating activity in a cultured human oral cancer cell line. Twenty 5-week-old male
NODscid mice underwent daily HBO2 of 2.5 atm abs, 90 minutes for 20 treatments. The control group, n =
20, did not undergo HBO2 and tumor weight, apoptosis index, and proliferating activity parameters were
compared between the two groups. The results showed no significant differences (p < 0.05) in the
whole-body weights, tumor weights, apoptotic index or proliferating activity index between the two groups.
By using the apoptosis and proliferating activity assays which were better indicators of tumor cell growth
than tumor weight alone, our results suggest that the clinical application of HBO2 does not promote the
growth or proliferation of human oral cancer cells.
INTRODUCTION
Hyperbaric oxygen therapy (HBO2) is presently one of the most effective adjunctive
treatments for osteoradionecrosis (ORN) (1-4). By increasing tissue oxygenation and
neovascularization, HBO2 promotes wound healing in irradiated tissues. Recent discoveries of
beneficial cellular biochemical effects have further strengthened the rationale for the clinical
application of HBO2 (5-10). However, concomitant malignancy has been recognized as a
contraindication to HBO2 because of possible growth enhancing effects to the tumor cells (11,
12). For patients with mandibular radionecrosis the most common scenario is that they undergo
surgery and radiotherapy to eliminate the cancer cells. After radiotherapy, soft tissue breakdown
and mandible erosion may develop. It is well known that HBO2 is helpful for patients with
wounds in radiated tissue but concern for residual and recurrent disease has made the timing of
HBO2 uncertain. The question of whether to use HBO2 in patients with ORN when there are
concerns about oral cancer is important. The purpose of this study was to determine whether the
cancer promoting effects of HBO2 occur in human oral cancer cells. It has been suggested that
possible cancer growth enhancing effects of HBO2 could be categorized by tumor nourishment,
immune suppression, and free radical-mediated toxicity (11). The traditional ways of evaluating
tumor growth include measuring the weights and/or the volumes of the tumors (13-15). Recently,
two more sensitive approaches, apoptosis and proliferating cell nuclear antigen (PCNA) assays,
have been applied in oncology studies (16-20). We used both approaches along with the
traditional tumor weight measurements to follow tumor progression and aspects of tumor
nourishment and toxicity during the application of HBO2.
Copyright © 2004 Undersea and Hyperbaric Medical Society, Inc. 251
UHM 2004, Vol. 31, No. 2 – HBO2 on human oral cancer cells
METHODS
Animals
Forty male non-obese diabetic/severe combined immune-deficient (NODscid) mice at 5
weeks old were used in this study. The immune competence of NODscid mice is decreased due
to poor T cell, B cell, and natural killer cell activity that allows human xenografts to be
transplanted successfully (21). Since NODscid mice are more immune-deficient than nude
athymic mice, a new trend in live culture media has developed for research (22). The mice
were housed at the Laboratory Animal Center of the Tzu Chi University, Taiwan. The Ethics
Committee for Animal Experimentation of the Tzu Chi University and Hospital approved the
experimental protocols.
Cell Cultures
An oral cancer cell line, CRL-1623, derived from human squamous cell carcinoma of the
tongue was provided by the American Type
Culture
Collection
(ATCC).
The
tumorigenecity of CRL-1623 is very high, it
is not too demanding to maintain and
subculture this cell line (23, 24), and the
40kD keratin filament by-product may be
used as an indicator to verify its squamous
cell origin (25). The cell line was propagated
in a mixture of 45% Dulbecco's modified
Eagle's medium, 45% Ham's F12 medium,
10% fetal bovine serum, and 400 ng/ml
hydrocortisone in a 75 cm cell culture flask
and incubated in saturated humidity at 37 °C
and 5% CO2 in air. The complete culture
medium was changed every 3 to 4 days. The
cells were subcultured with 0.25% trypsin,
0.03% ethylenediamine tetraacetic acid
(EDTA) solution after an adherent monolayer
was formed in the flask (Figure 1).
Fig. 1. Representative view of the adherent monolayer of the CRL-1623
human tongue squamous cell carcinoma cell line in a 75 cm2 culture flask. (400X magnification).
The cells were harvested after eight culture passages. Unit doses of 0.15 ml containing
106 single cell suspensions were prepared for subsequent inoculations of immunocompromized
experimental animals.
In vivo Tumor Xenografts
The NODscid mice were divided into to two groups randomly and raised in sterilized
cages with a density of five mice per cage. Each animal had access to sterile water and food ad
libitum. A 12-hour light and 12-hour dark cycle was performed automatically. The cultured
cancer cells, 106 cells in 0.15 ml phosphate buffered saline, were inoculated into the back of each
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mouse subcutaneously.
treatment.
All 40 mice were allowed to recover for 72 hours before HBO2
Hyperbaric Oxygen Treatment
A SIGMA-34 monoplace hyperbaric chamber (Perry Baromedical Corporation, Fla.,
USA) was used for this study. The study group, n = 20, received daily HBO2 from 10:20 a.m. to
12:20 p.m. Monday through Friday. The compression was started after 10 minutes of rest and 2.5
atm abs was reached within 10 minutes. During the treatment, the animals breathed the chamber
atmosphere, which was 100% O2. The pressure was kept at 2.5 atm abs for 90 minutes. For
decompression, the pressure was decreased 0.1 atm abs min-1 until 1 atm abs was reached. Each
animal underwent 20 treatments and all were completed smoothly without adverse effects. The
control group, n = 20, experienced the same transportation procedure simultaneously and were
treated with air at 1.0 atm abs.
Tumor Evaluations
After completion of the HBO2 treatments, the animals were euthanized with CO2. Surgical
dissections of the tumors were performed immediately after death (Fig. 2). An electronic scale was
used to measure the weight in milligrams of each tumor in the fresh state. The tumors were
subsequently fixed with buffered 10% formaldehyde for more than 72 hours. Blocks of tumors in
size of 0.5 × 0.5 × 0.5 cm3 were dehydrated using a graded
ethyl alcohol series and then infiltrated with liquefied paraffin.
The paraffin blocks were sliced to sections of 5 to 10 microns
in thickness with a sharp steel knife in the microtome. The
sections of tissue slices were placed on slides for Hematoxylin
and Eosin (H&E) stain, apoptotic assay, and proliferation assay.
Fig. 2a. A subcutaneous tumor derived
from CRL-1623 cells inoculated onto
the back of a NOD-SCID mouse.
Figure 2b (right). Dissection of CRL-1623 cell line-derived
xenografted tumor from subcutaneous layer in a NODscid
mouse. The firm yellowish tumors were well-defined,
highly-vascular, and adherent to the underlining muscular layer.
The H&E stain was used to confirm the morphologic characters of the tumor cells (26).
DNA fragmentation detection kits (TdT-FragEL) from Oncogene Research Products (Mass.,
USA) were used to detect the apoptosis of tumor cells by fragment end labeling of the DNA. In
this assay, terminal deoxynucleotidyl transferase (TdT) binds to exposed 3’-OH ends of DNA
fragments and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides during
incubation at 37°C for 1.5 hours. Biotinylated nucleotides were detected using a
streptavidin-horseradish peroxidase conjugate. The labeled sample was incubated with
diaminobenzidine at room temperature for 10 to 15 minutes to generate an insoluble colored
substrate at the site of DNA fragmentation. The specimen was counterstained with methyl green
as a contrast agent for morphological evaluation and characterization of normal and apoptotic
cells (27, 28). A positive control was generated by TdT-FragEL staining of human acute myeloid
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leukemia HL60 cells induced to undergo apoptosis with 0.5 µg/ml actinomycin D (slide supplied
by Oncogene, Mass., USA).
The proliferating activity of the tumor cells was evaluated using an immunohistochemical
stain. After endogenous peroxidase activity was quenched by incubating the specimen with 3%
hydrogen peroxide at room temperature, it was incubated with the primary mouse monoclonal
antibody (generated by immunizing BALB/c mice with recombinant PCNA fused with myeloma
SP2/0-Ag14; DAKO Inc, Calif., USA) overnight in a humidified chamber at 4o C. Then the
specimen was incubated with a biotinylated secondary anti-mouse antibody and
peroxidase-labeled streptavidin (DAKO Inc, Calif., USA). Staining was complete after 10
minutes of incubation with diaminobenzidine, which resulted in a brown precipitate at the
antigen sites. The background was counterstained with hematoxylin (29, 30).
Analysis and Statistics
Representative slides of H&E, TdT-FragEL, and PCNA assays for each tumor were
analyzed using a light microscope (Olympus, CH30, Tokyo, Japan) at magnifications of 400X
(H&E, TdT-FragEL) and 1000X (PCNA). Indexes of the apoptosis (TdT-FragEL stained cells)
and tumor cell proliferating activity (PCNA stained cells) were calculated using the formula:
total number of positive stained cells in 10 microscopic fields
10
All of the measurement values were expressed as mean ± SEM. Two-tail Student’s t-test
was performed for comparison between the experimental group and the control group. The
statistical significance was defined as P < 0.05.
RESULTS
Human oral cancer cells derived from the CRL-1623 cell line were inoculated into the
back of 40 NODscid mice successfully. By the end of the experiment, each animal had a
noticeable bulge in the inoculation region (see Figure 2, previous page). No seizures or other
hyperoxia-related complications developed during the experiment. The mean body weight of the
experimental group (32.2 ± 0.58 g) showed no significant difference between that of control
group (31.4 ± 0.67 g) at the end of the experiment.
The mean fresh tumor weight of the HBO2 treated mice was 4.01 ± 0.40 g, which
compared to the mean fresh tumor weights of the control animals (4.42 ± 0.44 g), was not
statistically different (Table 1). Microscopic examination of the control and experimental H&E
stained paraffin-embedded tumor sections revealed well differentiated squamous cell carcinomas
that were not morphologically distinct. Furthermore, the grades of differentiation of the tumors
from both the control and experimental animals were similar (Figure 3).
In addition to the static parameters of tumor growth, the apoptotic index and proliferating
activity of each tumor were examined. The functional apoptotic status of the tumor cells was
evaluated using the TdT-FragEL stain. There was very little apoptotic activity for the tumor
derived from the CRL-1623 cell line (Figure 4).
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Table 1: The effect of HBO2 on the CRL1623 cell line-derived xenograft tumors.
HBO2 group (n = 20)
Control group
(n = 20)
P value
Body Weight (g)
32.2 ± 0.58
31.4 ± 0.67
0.3844
Fresh Tumor Weight (g)
4.01 ± 0.40
4.42 ± 0.44
0.4954
Apoptotic Index
1.18 ± 0.26
1.23 ± 0.30
0.9030
Proliferating Activity Index
81.5 ± 2.77
84.8 ± 5.04
0.5485
Table 1. Values are expressed as mean ± SEM. Two tailed Student’s t-test was used for comparison of the data. The
apoptotic index is calculated from the mean positive TdT-FragEL stained cells of 400X light microscopy. The
proliferating activity index is calculated from the mean positive PCNA stained cells under light microscope with
1000X magnification. The body weight, fresh tumour weight, apoptotic index, and proliferating activity index of
CRL-1623 cell line-derived xenografted tumours in NODscid mice after 20 treatments with hyperbaric oxygen at
2.5 ATA for 90 minutes were all not significantly different from those in the control animals.
Fig. 3.
Representative
views of a
CRL-1623 cell
line-derived
xenografted
tumor in
NODscid mice.
The xenografted
tumor cells were
very similar to
those found in well-differentiated squamous cell carcinomas in human oral cavities. There is no difference in
morphology and grade-differentiation between HBO2-treated (left) and control (right) tumors (400X
magnification).
Fig. 4. Representative view of the apoptosis assay (TdT-FragEL)
in cells from CRL-1623 cell line-derived xenografted tumors in
NODscid mice. The arrow indicates a dark brown stained
apoptotic cell (400X magnification).
The calculated apoptotic index expressed as the
mean positive TDT-FragEL stained cells under 400X
light microscopy for the HBO2 treated group was 1.18 ±
0.26 and that for the control group was 1.23 ± 0.30.
There were no statistical differences in apoptotic activity between the two groups. The high
number of PCNA immunohistochemically-stained cells shown in Figure 5 indicates the high
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level of proliferating activity in the CRL-1623 cell line. The proliferating activity index for the
HBO2 group (81.5 ± 2.77) was not significantly different from that of control group (84.8 ±
5.04).
Fig. 5. Representative view of proliferating cell nuclear
antigen (PCNA)-stained cells from CRL-1623 cell
line-derived xenografted tumors in NODscid mice. The
arrows indicate the positive dark brown stained
proliferating cells. (400X magnification).
DISCUSSION
A growing body of strong evidence supports the use of HBO2 as an effective adjunctive
therapy for treating delayed radiation injury in various diseases (1-3, 31-37). Unfortunately, a
growth-promoting effect makes HBO2 a contraindication for patients with concerns of
malignancies (12). For example, many patients develop mandibular ORN after wide excision and
radiotherapy for oral cancers, and some who undergo painful surgical and radiation procedures
are found to have soft tissue break down and bony erosions months after their initial treatments.
Although it is clear that HBO2 is an effective adjunct for managing ORN, we considered the risks
of nourishment and proliferation of residual or recurrent tumor cells in a high oxygen
environment and/or the promotion of new malignancies due to ROS formation during HBO2
treatment of mandibular ORN to be worthy of further examination.
Feldmeier et al. reviewed 24 scientific articles published between 1960 and 1993. The
results of three clinical studies and two animal model studies supported the statement that HBO2
had a growth promoting effect on malignancies (11). However, most of the studies did not focus
on the oral cancers. One study on oral cancer used an animal model in which the buccal cancer
was induced by repeated application of the carcinogen dimethylbenzanthracene (DMBA) to the
buccal pouch of golden Syrian hamster (13). However, the experimental model in hamsters is far
removed from the human condition and the behavior of induced cancer in this model would not
be the same as for humans. Thus, the responses achieved during their study should not be used as
the final word (4, 11). For example, human papillomavirus type 16 and 18 demonstrate
oncogenicity by transforming normal human oral keratinocytes in vitro. However, repeated viral
inoculation of hamster oral mucosa failed to produce tumors or histopathological evidence of
malignancy (38). Marx and Johnson designed an investigation using the hamster cheek pouch
model (39). In contrast to the results of McMillan et al. (13) which suggested HBO2 had a
stimulatory effect during the proliferate phase of DMBA-induced carcinoma, their results
indicated that HBO2 had no effect on the development of early dysplastic changes and had a
suppressive effect on the transformation from dysplasia to malignancy. To elucidate the effects of
HBO2 on oral cancer, further studies using different models are necessary. Myers et al. used an
experimental animal xenograft to evaluate the effects of target molecular therapy for oral cancer
(40). However, HBO2 studies using xenograft animal models usually relied on static weight and
volume of tumors as parameters for evaluating tumor growth (13-15). The dynamic status of
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tumor cells including the number of cells programmed to die and the number of cells undergoing
proliferation remains unknown.
Apoptosis, or programmed cell death, has been a prevalent topic of research in recent
years (41-44). There are three different mechanisms by which a cell commits suicide via
apoptosis (45-48). First, apoptosis is triggered by death signals, either extrinsic or intrinsic.
Second, apoptosis is triggered by deprivation of survival signals. Third, apoptosis is induced by
specific radiation or anti-cancer drugs. Serial enzymatic reactions are induced and lead to
chromatin condensation and fragmentation of DNA (49). In this study, we used the terminal
deoxynucleotidyl transferase DNA fragmentation end-labeling technique to determine the status
of apoptosis for each tumor (50). PCNA is a cyclin protein of 37 kD molecular weight also called
polymerase-δ accessory protein. It is recognized that there are very high correlations between
PCNA expression and the proliferation activity of cells (51). Recently, PCNA has been used to
evaluate tumor growth in various malignancies, including lung cancer, (52) hepatocellular
carcinoma, (19, 20) squamous cell carcinoma, (53) and tumors originating in the central nervous
system (18). In the present study, immunohistochemical staining for PCNA was used to indicate
the proliferating activity of tumors derived from a human oral cancer cell line. To our knowledge,
there has been no previous HBO2 study that used the apoptosis and PCNA assays to evaluate the
HBO2 associated cancer-promoting effects. Most tumor evaluation methods have used tumor
numbers, weights, and volumes. It has been recognized that the PCNA labeling index correlated
with the clinical outcome and grading of malignant central nervous and hepatic tumors (18, 19).
Greene et al. used apoptosis and PCNA assays to evaluate the effects of basic fibroblast growth
factor (bFGF) and the angiogenesis inhibitor TNP-470 on hepatic regeneration after partial
hepatectomy. Their results suggested that the cessation of the regenerative process correlated
with a decrease in endothelial proliferation and an increase in endothelial apoptosis (54). Kohno
et al. also demonstrated that the apoptosis and PCNA labeling index reflected the pathological
alterations of human oral carcinogenesis (55).
It is clear that the application of the apoptosis and PCNA assays provided evidence about
the dynamic nature of HBO2 on human oral cancer cells. The in vivo xenograft model used in
our study presented a very hypercellular picture. Roughly, an average of 1076 tumor cells was
seen in a microscopic field of 400X magnification. Comparing with the data of Kohno et al., in
which the experimental oral tumors presented a 35% positive stain for PCNA and 22% for
apoptosis assay (55), our data demonstrated a very active proliferation activity (about 80%
positive stain for PCNA) and extremely low apoptosis (about 1%). The results of these two novel
assays in our study showed that the malignant tumors derived from the CRL-1623 cell line of
both the HBO2 group and the control group had low apoptotic and high proliferation potentials.
HBO2 did not show significant effects on toxicity or nourishment of these squamous carcinoma
cells. Most of the previous studies involved assessment of cancer recurrence over a short period
of time. It was not realistic for HBO2-mediated cancers to recur on a long time scale following
treatment.
An advantage of this study using apoptotic and proliferating activity indices was that
these assays may potentially indicate growth recurrence of squamous cell carcinoma over a
longer time scale. For the low apoptosis index shown in our results, a positive control was
established by using TdT-FragEL staining of human leukemia HL-60 cells induced to undergo
apoptosis with 0.5 µg/ml actinomycin D to verify the apoptosis assay functioned properly. We
did not use a second cell line to examine for differences in apoptotic activity index in our animal
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model. However, new evidence on the benign effects of HBO2 was demonstrated on oral cancer
cells.
In summary, we used NODscid mice with profound immunodeficiency, CRL-1623 cell
line of high and constant tumorigenecity, and the dynamic evaluation of tumor growth using
TdT-FragEL and PCNA assay. The results showed that there were no significant differences in
body weight, tumor weight, apoptotic index or proliferating activity between the HBO2 group
and the control group.
CONCLUSION
The results of our study showed that 20 HBO2 treatments at a dose of 2.5 atm abs for 90
minutes did not promote the growth or proliferation of human oral cancer cells.
ACKNOWLEDGMENTS
This work was supported by Buddhist Tzu Chi Medical Center grant TCRD91-21. The
authors gratefully acknowledge the assistance of Miss Tsuey-Hwa Tsai, Prof. Terry B.J. Kuo,
Prof. Cheryl C.H. Yang, and Department of Medical Research.
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