Effects of ionizing radiation on the viability and proliferative behavior of the human glioblastoma T98G cell line
Murad et al. BMC Res Notes
Effects of ionizing radiation on the viability and proliferative behavior of the human glioblastoma T98G cell line
Hossam Murad 0
Yaman Alghamian 2
Abdulmunim Aljapawe 0
Ammar Madania 1
0 Human Genetics Division, Department of Molecular Biology & Biotechnology, Atomic Energy Commission of Syria (AECS) , P.O. Box 6091, Damascus , Syria
1 Department of Radiation Medicine, Atomic Energy Commission of Syria (AECS) , Damascus , Syria
2 Department of Animal Biology, Faculty of Sciences, Damascus University , Damascus , Syria
Objective: Radiotherapy is the traditional therapy for glioma patients. Glioma has poor response to ionizing radiation (IR). Studying radiation-induced cell death can help in understanding the cellular mechanisms underlying its radioresistance. T98G cell line was irradiated with Co60 source by 2 or 10 Gy. MTT assay was used to calculate the surviving fraction. Cell viability, cell cycle distribution and apoptosis assays were conducted by flow cytometry for irradiated and control cells for the 10 Gy dose. Results: The SF2 value for irradiated cells was 0.8. Cell viability was decreased from 93.29 to 73.61%, while, the Sub G0/G1 phase fraction was significantly increased at 10 Gy after 48 h. On the other hand, there was an increase in the percentage of apoptotic cells which reached 40.16% after 72 h at the same dose, while, it did not exceeds 2% for nonirradiated cells. Our results showed that, the T98G cells is radioresistant to IR up to 10 Gy. Effects of irradiation on the viability of T98G cells were relatively mild, since entering apoptosis was delayed for about 3 days after irradiation.
Glioma; T98G cell line; Ionizing radiation; Radioresistance; MTT; Flow cytometry; Apoptosis
Glioblastomas are tumors affecting the central
nervous system. They rank among the most common
primary tumors worldwide [
]. Only a small percentage
of patients live for 2 years after disease onset [
tumors are traditionally treated by surgery, however, it is
impossible to completely eradicate the tumor due to its
penetrating nature with neighboring tissues [
Therefore, surgery is followed by radiation therapy (60 Gy
total dose within 6 weeks), which increases the chance of
recovery. In most cases, radiation resistant cancer cells
settle in sensitive locations of the brain, which cannot be
accessed by surgery, leading to relapse [
Several studies investigated cellular behavior after
exposure to IR, the capacity of radiation to stop
proliferation of cancer cells and to induce apoptosis [
induces DNA damage, this damage can influence cell
]. After DNA damage occurs, DNA damage
pathways are activated, leading to cell cycle arrest, DNA
damage repair, cell proliferation, senescence, or apoptosis
]. Exposure to IR causes failure in cell division and
loss of some genetic material, showing an aberrant
distribution of chromosomes during division [
studies showed that, preferential activation of the DNA
damage checkpoint and enhanced DNA repair capacity
in gliomas lead to radioresistance [
9, 14, 15
depending on targeting DNA damage response network
in gliomas were applied to sensitize tumors and reverse
]. Furthermore, another
studies showed abrogating the cell cycle checkpoint could
increase radiosensitivity in glioma cell line [
Several studies showed that IR causes glioma cells to
enter apoptosis [
]. Apoptosis is an active
mechanism of cell death, requiring the activation of a cascade
of caspases genes, leading to DNA fragmentation and
plasma membrane disintegration . It has been shown
that the extent of this process determines cells
sensitivity to IR [
]. Some studies have shown that radiation did
not cause apoptosis in the U343 cell line derived from
glioblastoma multiforms, which makes it radioresistant
]. Full understanding of the glioblastomas response
to IR and detailed radioresistance analysis may help to
identify radiosensitizing agents of this fatal disease.
Several methods have been developed to measure cell
sensitivity to radiation, such as the MTT
spectrophotometric assay [
]. The SF2 value (surviving fraction at
2 Gy) is used as an indicator for cell sensitivity to
radiation. Cells are considered radioresistant if the SF2 value
exceeds 0.5 and radiosensitive if the SF2 value is less than
Flow cytometry has been used to determine cell
viability and rate of cell death caused by radiation in cell lines
derived from glioblastomas [
]. Flow cytometry
can also be used to study cell proliferation and cell cycle
distribution utilizing cellular DNA content histograms.
Annexin-V assay is a quantitative method that can be
used to detect and quantify cells entering apoptosis [
The aim of this study was to estimate the sensitivity of
the T98G cells (as a model of glioblastomas) to IR and to
investigate the effects of a high dose of IR on the T98G
cell lines at the cellular levels, by studying the cell
viability, cell cycle and apoptosis to investigate the behavior of
this cells to this dose.
Materials and methods
T98G cells were kindly provided by Prof. Dr. P. Bécuwe,
University of Nancy. Sub cultures of this cell line were
maintained in complete RPMI-1640 medium containing
10% FBS (fetal bovine serum), 2 mM glutamine, 0.1 mg/
ml each of penicillin and streptomycin, at 37 °C and 5%
CO2. All cell culture media were purchased from (Gibco,
Cultures of T98G cells were irradiated in the
irradiation reference laboratory at (AECS) using a Co60 source
(Theratron 80, USA), Doses used were 2 or 10 Gy and the
dose rate was of 447 mGy/min.
MTT spectrophotometric assay
The MTT kit (Roche, Germany) was used as previously
] to measure T98G cells sensitivity to
radiation. Cells were cultured in 96-well plates (5000 cells/
well) and irradiated with the 2 Gy. The value of SF2,
which reflects the sensitivity of the cells to radiation,
was calculated using the following equation: SF = 2 − (t
delay/t doubling time), where: “t delay” is the time
needed for irradiated cells to reach an absorbance equal
to that of control cells, “t doubling time” is the time cells
need to double their number.
Cell cycle analysis
T98G cells were treated with a 10 Gy of IR and returned
to the incubator for 6, 24, 48 and 72 h. Cells were
collected, washed with PBS and stained for cell cycle analysis
using BD Biosciences cell cycle test kit (BD Biosciences,
USA). For each sample, 1 × 104 cells were analyzed using
the BD FACSCalibur flow cytometer (Becton–Dickinson,
Cell viability assay
T98G cells were irradiated with the 10 Gy, then,
harvested after 6, 24, 48 and 72 h. Cells then washed with
PBS buffer and stained for cell viability using BD
Biosciences viability kit.
10 Gy irradiated T98G cells were collected after 6, 24,
48 and 72 h of irradiation. Cells then washed with PBS
buffer and stained using BD Biosciences Apoptosis
Detection Kit I.
The SPSS 23 software (SPSS, Chicago) was used in
statistical analysis. The Student-t test was applied to analyze
the differences between treatments. Differences were
considered statistically significant at *P < 0.05.
Radiosensitivity and viability of T98G cells
The SF2 value for cells irradiated with 2 Gy was 0.8,
which is clearly greater than 0.5, indicating that the T98G
cells are radioresistant. As shown in (Fig. 1), growth of
irradiated cells was delayed about 12 h compared to
nonirradiated cells. Viability of T98G cells exposed to a 10 Gy
was dropped to 93.29, 91.62 and 73.61% after 6, 24 and
48 h respectively, (Fig. 2a).
Effect of IR on the cell cycle of T98G cells
As shown in Fig. 2b, the percentage of dead cells
increased to 3.53, 3.43, 7.93 and 13.3% after 6, 24, 48 and
72 h of irradiation respectively. We found that the
percentage of cells found in G1 phase was decreased after 6,
24, 48 and 72 h to 73.64, 63.29, 49.52 and 46.97%
respectively, after irradiation with 10 Gy. While the
percentage of 10 Gy irradiated cells found in G2 phase was 9.22,
22.11, 26.33 and 22.66% after 6, 24, 48 and 72 h
respectively showing a slight G2/M cell cycle arrest.
Effect of IR on apoptosis of T98G cell line
We used the double staining method (annexin V-FITC
and IP) and flow cytometry to determine the percentage
quadrant). Flow cytometric analysis demonstrated that
after irradiation with 10 Gy, apoptosis rate (sum of the R1
and R3 quadrants) increased from 9.63 to 20.88% and to
40.16% after 24, 48 and 72 h respectively.
Glioblastomas represent one of the deadliest cancer
types, where affected patients generally die within 2 years
after disease onset [
]. In spite of the high
radioresistance of glioblastoma cells, IR remains one of the
traditional therapies for those tumors [
of cancer cells was the subject of numerous studies, due
to its importance in cancer therapy practice and
implications in several molecular pathways, such as DNA repair,
cell cycle check points and cell death [
14, 36, 37
high resistance of glioblastoma cells to radiotherapy is
attributed to weak entrance into programmed cell death
induced by IR . Ionizing radiation induces damage to
the genetic material of the cell, negatively affecting
several vital cellular mechanisms [
]. As a response to these
damages, cells can select one of several possible pathways
according to the nature, intensity and duration of the
induced effect (chemotherapy, radiotherapy,
pharmacological drugs, etc.) [
]. Cells can continue their
division, ignoring the induced effect, or their cell cycle arrest
of cells undergoing programmed cell death due to
irradiation. As shown in Fig. 3, we distinguished four groups of
cells: live (annexin V− PI−, R2 quadrant), early apoptotic
(annexin V+ PI−, R3 quadrant), late apoptotic (annexinV+
PI+, R1 quadrant) and necrotic (annexin V− PI+, R4
until damages are repaired, then enter into senescence,
differentiate, or enter into apoptosis if the damages are
irreparable . Furthermore, the fate of the tumor cell
after radiotherapy is determined by its characteristics,
such as type and tissue of origin [
]. The SF2 value is
considered as an indicator for cells radiosensitivity [
]. Our results showed that, SF2 value was 0.8, so the
T98G cells were considered as radioresistant (Fig. 1).
Previous study by Liu et al. showed that, T98G cells had
SF2 value reach to 0.7 compared to other human cell
lines . In our study, the viability of irradiated cells
(up to 10 Gy) was slightly decreased after 48 h (Fig. 2a).
Furthermore, exposing T98G cells to a 10 Gy, (which is
considered relatively a high dose), induced a significant
cell death only after 72 h (Fig. 2b). Roy et al. showed that,
the sensitivity of the tow U118 and U87 cells
(glioblastoma cell lines) to IR at 6 Gy was more than (> 2-fold)
compared to T98G cells [
]. Another study by Yao et al.
revealed that, there was a significant inhibition of cell
proliferation at 20 Gy for (GB-1, T98G, U251-MG, and
U373-MG) cells at 24 h, and the apoptosis did not occur
in any these cells following irradiation [
]. Our results
showed, no accumulation of irradiated T98G cells in the
SubG0/G1 phase after a 10 Gy at 6, 24 and 48 h, whereas,
we observed a limited cell death at 72 h. Yao et al. did
not observe any dead T98G cells irradiated at 5 Gy in the
SubG0 phase after 4 days [
]. Our results showed that,
a 10 Gy irradiation resulted in a G2/M cell cycle arrest.
This phase gives the cells an opportunity to repair
damaged DNA induced by irradiation. However, when DNA
damage is irreparable cells undergo apoptosis [
9, 42, 43
In addition, double staining with annexin-V-FITC
and PI showed that, the rate of apoptotic irradiated
T98G cells reached 40.16% at 72 h. Ma et al. pointed
that, there was only 16% of death rate after 96 h for
U251 MG cells (glioblastoma cells) irradiated at 7 Gy
]. Several studies suggested a combination treatment
for T98G cells to improve their radiosensitivity to IR.
Tani et al. showed that, the continuous down-regulation
of γ-glutamylcysteine synthetase (γ-GCS) expression by
hammerhead ribozyme (as a potential anticancer gene
therapy), increased the cytotoxicity of the T98G cells to
]. Although, treatment of T98G cells with BI 2536
(as an inhibitor for Polo-like kinase 1) caused mitotic
arrest and increased apoptosis in irradiated cells after
24 h [
Taken together, our results showed that even in the
highdose, 10 Gy, cells did not respond to ionizing radiation
after 24 h, while, these cells needed about 72 h to inter in
apoptotic phase by a rate not exceeding 40% at the same
dose. We conclude that, the ionizing radiotherapy alone
even in a high dose, does not lead to the efficient
treatment in advanced grade glioblastoma patients.
Further studies are necessary in order to elucidate the
molecular mechanisms causing cell resistance to IR,
and to identify the genes responsible for radioresistance.
These genes would be potential targets for targeted
therapies that would improve response to IR and force cancer
cells to enter cell death.
SF2: surviving fraction at 2 Gy; MTT:
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide); TO: thiazole orange; PI: propidium iodide.
HM, YA and AA conceived the study, its design and coordination. AM drafted
the manuscript. All authors read and approved the final manuscript.
We would like to thank Prof. Ibrahim Othman, the Director General of AECS,
and the Head of department of Molecular Biology & Biotechnology and also,
the head of department of Radiation Medicine for their support.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published
Consent for publication
Ethics approval and consent to participate
This study has been approved by the Institutional Review Board of the Atomic
Energy Commission of Syria (AECS).
This project was financially supported by Atomic Energy Commission of Syria
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