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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 6  |  Page : 1273-1278

Cytogenetic damage from hyperthermia,6 MV X-rays, and topotecan in glioblastoma spheroids, simultaneously, and separately


1 Radiation Biology Research Center, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
2 Radiation Research Center, School of Allied Medicine, AJA University of Medical Sciences, Tehran, Iran
3 Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
4 Department of Radiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Web Publication28-Nov-2018

Correspondence Address:
Nazila Eyvazzadeh
No. 6, Shikh Fazlolah Building, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.189239

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 > Abstract 


Purpose: Glioblastoma multiform (GBM) is one of the most common brain tumors. Surgery, radiation therapy, hyperthermia, and chemotherapy are the most common treatments for brain tumors such as GBM. This study investigated the cytogenetic damage caused by hyperthermia, radiation (6 MV-X-rays), and topotecan in glioma spheroids, simultaneously and separately.
Materials and Methods: Human glioblastoma cell line was cultured to form spheroids 350 μm in diameter that were arranged in eight groups and coded as follows: control, T: topotecan, H: hyperthermia, T + H: topotecan + hypertermia, X 1–10: X-ray with 1–10 fraction irradiation, H + X (1–10): hypertermia + X-ray with 1–10 fraction irradiation, T + X (1–10): topotecan + X-ray with 1–10 fraction irradiation, and H + T + X (1–10): hypertermia + topotecan + X-ray with 1–10 fraction irradiation. DNA damage was then evaluated using clonogenic assay.
Results: The effect of combined treatment with X + H + T was greater than the sum of the effects in other groups. In H + T + X group, failure to form colonies was observed in the seventh session.
Conclusion: Use of X + H + T combination therapy significantly increased cell death and possibly improved the treatment. This suggests that the synergistic effect of different therapeutic methods increased cell death in glioblastoma tumor cells and reduced the necessary dose of radiation in the treatment of tumor in radiation therapy.

Keywords: Glioblastoma, hyperthermia, radiation therapy, topotecan


How to cite this article:
Neshasteh-Riz A, Eyvazzadeh N, Rostami A, Azzizolahi E. Cytogenetic damage from hyperthermia,6 MV X-rays, and topotecan in glioblastoma spheroids, simultaneously, and separately. J Can Res Ther 2018;14:1273-8

How to cite this URL:
Neshasteh-Riz A, Eyvazzadeh N, Rostami A, Azzizolahi E. Cytogenetic damage from hyperthermia,6 MV X-rays, and topotecan in glioblastoma spheroids, simultaneously, and separately. J Can Res Ther [serial online] 2018 [cited 2019 Sep 21];14:1273-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/6/1273/189239




 > Introduction Top


Glioblastoma multiform (GBM) is one of the most common and most deadly types of malignant brain tumors; it is a primary tumor, classified as a central nervous system in origin. GBM has a high prevalence among African and American people and is more commonly seen in patients aged 45–65-year-old.[1] Surgery followed by radiation therapy is the most common way to treat brain tumors such as GBM.[2],[3],[4],[5] Unfortunately, the dose of radiation at which the drug is effective for the treatment of GBM is much higher than the level of tolerance of normal brain tissue.[6] Therefore, it is important to identify treatments in which tumor cells are more sensitive to radiation to reduce the radiation dose required to treat them and thus avoid the adverse effects of radiation on normal brain tissue. One such method of treatment is the application of a combination of radiosensitizer and chemotherapy drugs with radiation therapy.[7] In addition to radiotherapy, another method used in cancer treatment in combination with radiation therapy for more effective treatment of tumors is the method of hyperthermia or heat therapy. This type of treatment involves raising the temperature of tumor tissue by microwave radiation, radio waves, and other methods to 45°.[8] Research in this field has shown that high temperature can cause degradation of intracellular proteins and shrink structures such as tumor cells.[9] Such treatment can also stimulate and speed up chemical reactions that lead to the death of tumor cells with minimum damage to normal tissue.[10] Hyperthermia, in addition to toxicity in cancer cells and induction of apoptosis, causes cells to be more sensitive to ionizing radiation.[11],[12]

Topotecan is a specific inhibitor of topoisomerase I that interferes with the processes of replication and transcription in tumor cells, eventually leading to cell death. The use of topotecan increases the sensitivity of exposure cells, as well as prevents damage restoration target cells through radiation.[13],[14] A particular feature of a topotecan, which has made it useful for the treatment of brain tumors, is that it can freely penetrate the blood-brain barrier.[15] Recent studies have shown that topotecan is most effective when used 2–4 h before irradiation, otherwise it does not have a significant effect.[16]

Despite the use of different methods such as surgery, radiation therapy, chemotherapy, and/or a combination of these methods, an effective and useful method for effective treatment and increased survival rate of patients with glioma type tumors remains to be determined.[7],[17] Thus, it is essential to investigate the potential for a more efficient and safer method to treat gliomas. This study investigated the cytogenetic damage caused by hyperthermia, radiation (6 MV-X-rays), and topotecan in glioma spheroids, simultaneously and separately.


 > Materials and Methods Top


Cell line

Human glioblastoma cell line U87MG was purchased from the Pasteur Institute of Iran and cultured in minimum essential medium (MEM; Gibco/Invitrogen, USA) that supplemented with 10% fetal bovine serum (FBS; PAA/Austria), 100 U/ml of penicillin-streptomycin (PAA/Austria) and 20 U/ml of Fungizone (Gibco/Invitrogen, USA).

Monolayer culture

Cells were cultured as a monolayer at a density of 104 cells/cm2 in T-25 tissue culture flasks (NUNC). Cultures were maintained at 37°C in a humidified atmosphere and 5% CO2. Cells were harvested by trypsinizing cultures with 0.25% trypsin and 0.03% ethylenediaminetetraacetic acid (EDTA) (Sigma) in phosphate buffer saline (PBS).

Spheroid culture

Spheroids were cultured using the liquid overlay technique. 5 × 105 cells were seeded in 100 mm T-25 flasks (NEST) coated with a thin layer of 1% agar with 10 ml of MEM supplemented with 10% FBS. The plates were incubated at 37°C in a humidified atmosphere and 5% CO2. Half of the culture medium was replaced with fresh culture medium every 3 days to feed the cells.

Growth curve

After three passages of monolayer culture, cells were cultured at a density of 10,000 per well in multiwell plates (24 wells/plate) (Greiner). The multiwell was incubated at 37°C in a humidified atmosphere and 5% CO2. For 9 days, at 24 h intervals, the cells from triplicate wells were removed by 1 mM EDTA/0.25% trypsin (w/v) treatment and counted in a hemocytometer. An average of nine counts was used to define each point (mean ± standard error of the mean [SEM]). Half of the culture medium was replaced with fresh medium twice per week. Then, the growth curve was plotted. In the linear area or logarithmic phase of the curve, the cells follow this equation:

N = N0 × ebt

Here, N0 is the initial number of the cells, N is the number of the cells after time t, and b shows the gradient of the logarithmic phase of the curve. Then, the population doubling time of the cells is determined according to the gradient of the logarithmic phase of the curve.

Cell preparation

Human glioblastoma cell line U87MG was cultured to form spheroids 350 μm in diameter that were arranged in eight groups and coded as follows: control, T: topotecan, H: hypertermia, T + H: topotecan + hypertermia, X 1–10: X-ray with 1–10 fraction irradiation, H + X (1–10): hypertermia + X-ray with 1–10 fraction irradiation, T + X (1–10): topotecan + X-ray with 1–10 fraction irradiation, and H + T + X (1–10): hypertermia + topotecan + X-ray with 1–10 fraction irradiation.

Drug treatment

Four of groups coding T, T + H, T + X (1–10), and H + T + X (1–10) treated with topotecan with a concentration of 1 μM in MEM containing 10% FBS 2 h before being exposed to hyperthermia and ionizing irradiation. After the treatment time, the medium containing drugs was removed, and the cultures were washed with PBS.

Hyperthermia and irradiation

Exponentially growing spheroids coding H, T + H, H + X (1–10), and H + T + X (1–10) were immediately immersed in a water bath (Memmert) maintained at a constant temperature of 43°C for an hour.

X-ray was administered by 6 MV linac (Varian 2100.CD) X 1–10, H + X (1–10), T + X (1–10), and H + T + X (1–10) groups with 2–20 Gy dose in 1–10 fractions at 37°C (temperature), 87.9 KPa (pressure), 100 cm SSD, and 10 cm2 field size. At the end of the exposure, the cell death was evaluated using the colony formation assay.

Clonogenic assay

Clonogenic assay is the method to determine cell death following radiation.[18]

The clonogenic ability and surviving fraction of GBM cells were evaluated by the colony assay according to the manufactured protocol and as previously described by Franken et al.[18] After the treatment was carried out on cells of each group, cells are seeded out in appropriate concentrations into 25 cm2 flasks. Colonies were fixed with formaldehyde 0.2% (v/v) for 5 min, stained with crystal violet (0.5% w/v) for 40 min, and counted using an optical microscope after 11 days. Colonies were defined as cells aggregation approximate number of which was more than 50. A number of colonies, plating efficiency (PE), and survival fraction (SF) were calculated. The PE is the ratio of the number of colonies to the number of cells seeds. The PE was calculated based on the above definition. Clonogenic efficiency is shown as the SF, which is described as PE in treated cells divided by PE in untreated cells accordance with the formula. At the end, the SF was calculated after determining PE.

Statistical analysis

Statistical analysis was performed using independent samples t-test and one-way analysis of variance followed by Scheffe test as the post hoc analysis using the SPSS version 16 (IBM company, United States). The value of P < 0.05 was considered to be significant. All values are expressed as mean ± SEM for all tests.


 > Results Top


The U87MG cells were able to form spheroids in liquid overlay cultures and show the phase contrast micrographs of these spheroids in 350 μm diameter on day 24 [Figure 1].
Figure 1: Plating efficiency for glioblastoma multiform cell line in control, topotecan, hypertermia, hypertermia + topotecan, X1, hypertermia + X1, topotecan + X1, and hypertermia + topotecan + X1 groups. Data are presented as mean ± standard error of the mean

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The volume doubling time of these spheroids was approximately 63.49 ± 0.75 h which was applied as drug treatment time consequently.

Determination plating efficiency and survival fraction

An average number of colonies and PE for GBM cell line in the control group are shown in [Table 1]. Maximum PE was observed when 5000 cells were seeded into flasks (3.62%). The fraction of cell survival and PE in groups that treated with 2 Gy dose irradiation (single dose), hyperthermia, and topotecan simultaneously and separately in the compare control group are shown in [Figure 1] and [Figure 2].
Table 1: Average number of colonies and plating efficiency for glioblastoma multiform cell line in control group

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Figure 2: Fraction of cell survival for glioblastoma multiform cell line in control, topotecan, hypertermia, hypertermia + topotecan, X1, hypertermia + X1, topotecan + X1, and hypertermia + topotecan + X1 groups. Data are presented as mean ± standard error of the mean

Click here to view


The fraction of cell survival and PE in groups that irradiated in 10 fractions (each fraction: 2 Gy) and treated with hyperthermia and topotecan simultaneously and separately in the compare control group are shown in [Figure 3] and [Figure 4].
Figure 3: Plating efficiency for glioblastoma multiform cell line in X-ray with 1–10 fraction irradiation, hyperthermia + X-ray with 1–10 fraction irradiation, topotecan + X-ray with 1–10 fraction irradiation, and hyperthermia + topotecan + X-ray with 1–10 fraction irradiation groups. Data are presented as mean ± standard error of the mean

Click here to view
Figure 4: Fraction of cell survival for glioblastoma multiform cell line in X-ray with 1–10 fraction irradiation, hyperthermia + X-ray with 1–10 fraction irradiation, topotecan + X-ray with 1–10 fraction irradiation, and hyperthermia + topotecan + X-ray with 1–10 fraction irradiation groups. Data are presented as mean ± standard error of the mean

Click here to view


During a 10-day treatment protocol with 2 Gy per day by 6 MV linac (Varian 2100.CD) between different groups in consecutive therapy sessions, statistically significant difference was observed in ability for colony formation [P < 0.05, [Figure 3].

As shown in [Figure 4], failure to form colonies in the X group was observed in the tenth session; also a 12–10% decrease in survival was observed in each session. In H + X group, failure to form colonies was observed in the ninth session; also there was a 10–15% decrease in survival observed in each session. In T + X group, failure to form colonies was observed in the ninth session; also a 12–20% decrease in survival was observed in each session. In H + T + X group, failure to form colonies was observed in the seventh session; also a 20–30% decrease in survival was observed in each session.

[Table 2] shows an increase in cell death percentage 300 μm spheroids in groups of X, H, T, and X + H + T in comparison to control group. The effect of combined treatment with X + H + T was greater than the sum of the effects in three groups of X, H, and T (P < 0.05).
Table 2: Increases in cell death percentages in U87MG spheroids in groups of X, hypertermia, topotecan, and X + hypertermia + topotecan in comparison to control group

Click here to view



 > Discussion Top


This study investigated the cytogenetic damage caused by hyperthermia, radiation (6 MV-X rays), and topotecan in glioma spheroid cells, simultaneously and separately.

Hyperthermia, in addition to toxicity in cancer cells and induction of apoptosis, makes cells more sensitive to ionizing radiation.[11],[12]

In hyperthermia treatment, it is important that heat distribution is uniform and homogeneous throughout the volume the tumor, this is achieved in most in vitro studies by using a water bath to raise the temperature of the cells up to 45°C. According to other research, the best condition to apply hyperthermia on glioblastoma cells is reportedly 43°C for 1 h.[11],[12],[19],[20],[21] The best interval between hyperthermia and radiation has been estimated from 1 to 6 h after heating.[22] In this study, spheroid glioblastoma cells were exposed to hyperthermia for 1 h in a water bath 43°C, and cells were irradiated with radiation (6 MV-X-rays) 1 h after hyperthermia treatment. In hyperthermia groups: (H + X) and (H + T: Topotecan), glioma cell death was significantly higher than in groups that were not treated with hyperthermia: (X) and (T) alone (P < 0.05). Cell death for (H + X) was higher than that of (H + T), which indicates that the maximum synergistic effect in glioma cell death between the groups was observed in (H + X). The results of this study were consistent with results of previous studies.[21],[23],[24]

Man et al. reported that application of hyperthermia before radiation in glioma cells served to reduce activation of survival kinase Akt and impaired glioma cell proliferation and so that these cells were more sensitive to radiation.[25]

Niedbala et al. report the combined radiation with hyperthermia in the treatment of human breast carcinoma introduced hyperthermia as a strong radiosensitizer.[21]

Hervault et al. observed that application of hyperthermia in combined pharmaceutical agents increased cell damage compared to the use of only pharmacological agents, and hyperthermia caused more transfer of the drug to tumor cells.[24]

Topotecan is an effective chemotherapy drug that interferes with strand break repair process of DNA and causes stabilization of damage through inhibition of topoisomerase I enzyme, so it is a good option for fixing damage caused by radiation therapy and cell death induction.[3],[8],[21] In topotecan groups: (TPT + X) and (TPT + H), glioma cell death was significantly higher than in groups without topotecan: (X) and (H) alone (P < 0.05). Cell death in the group (TPT + X) was higher than that in (TPT + H), which indicates that the maximum synergistic effect in glioma cell death was between the groups mentioned in the synergistic effect of topotecan and X-ray radiation (H + X). The results of this study were consistent with those of the previous studies.[23],[26],[27]

Our results revealed that a single dose of 2 Gy X-ray irradiation after treatment with topotecan and apply hyperthermia caused a 32% increase in cell death in glioblastoma cells compared to the control group.

Fractionation radiation was used to simulate cell studies, as well as clinical studies in addition to single-dose radiation. Results revealed that 20 Gy X-ray radiation during the 10-day fractionation protocol resulted in the lowest rate of survival at each fraction of treatment protocol in (X-H-TPT) compared with (X-H), (X-TPT), and (X). Stop in the ability to form colonies in (X-H-TPT) group was observed in the seventh session, while colony formation failed for H + X, T + X, and X in the ninth and tenth sessions, respectively. This means that integration of damage caused by radiation, hyperthermia, and topotecan reduced the number of sessions of radiotherapy necessary for cellular death. In addition to these results, it is suggested that the synergistic effect of simultaneous methods of treatment; hyperthermia, topotecan, and X-ray to cause cell damage, and death is more effective than each treatment used alone. In addition, by increasing the amount of injury necessary to stop the formation of colonies in an (X-H-TPT) fractionated dose decreased compared to doses in other groups. In this way, by reducing the necessary radiation dose to create cell damage in glioblastoma cell presents a major step toward improving the treatment of this type of tumor.


 > Conclusion Top


The use of X + H + T combination therapy significantly increased cell death and possibly improved treatment. This suggests that the synergistic effect of different therapeutic methods increased cell death in glioblastoma tumor cells and reduced the radiation dose necessary to treat tumors in radiation therapy. Moreover, in addition to reducing treatment time, the synergistic effect of different therapeutic methods can decrease damage to normal tissue around the tumor tissue. For this reason, it is recommended effectiveness of different chemotherapy drugs with various doses and various forms are evaluated in combinations of different therapeutic methods such as hyperthermia and radiation therapy.

In future, a study is needed to evaluate the simultaneous effects of chemotherapy drugs with polymeric nanoparticle shape in combination with hyperthermia and radiotherapy. Chemotherapy drug administration polymeric nanoparticles can increase the drug transfer to the cells and increase the effectiveness of hyperthermia treatment.

Financial support and sponsorship

This research is supported by Iran University of Medical Sciences Grant.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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