1. Introduction
adiation therapy is one of the standard cancer therapies. It can be used alone or in combination with chemotherapy. Radiotherapy aims to kill maximum cancer cells with minimal damage to healthy tissue. Radiation resistance and the inherent flaws of the therapeutic system create a balance between its therapeutic advantages and physiological disadvantages. Multiple approaches have been used to enhance its efficacy while reducing toxicity. These approaches include (I) enhancing radiosensitization of tumor tissue, (II) reversal of radiation resistance in tumor tissue, and (III) enhancing radioresistance of the healthy tissue [1]. Clone cancer is the fourth leading cause of cancer death [2]. Ionizing radiation is known as a factor that induces micronucleus formation in cells. Increased micronucleus formation has also been observed following many radiosensitizing compounds in cells [1]. Numerous studies show the ability of many anti-inflammatory agents, especially nonsteroidal anti-inflammatory drugs (NSAIDs), in inhibiting tumor growth [3, 4, 5]. Tolmetin is an NSAID that inhibits the synthesis of prostaglandins. The regulatory effect of tolmetin on anticancer drugs has already been studied. Tolmetin and other NSAIDs increase the toxicity of anticancer drugs by the inhibition mechanism of ß-catenin [6, 7]. Therefore, the present study aimed to evaluate the effect of radiosensitization of tolmetin in radiotherapy treatment on the HT-29 colon cancer cell line. 
2. Methods
Human clone cancer cell line HT-29 (ATCC HTB-38) was purchased from the National Center for Biological and Genetic Resources of Iran. Cells were cultured in RPMI (Dacell) culture medium, containing 10% fetal bovine serum (Gibco), streptomycin 200 μg/mL, and penicillin 500 U/mL (Gibco). Finally, they were kept in an incubator at 37°C, under 5% CO2 and 95% humidity. Exponentially growing HT-29 cells were treated with tolmetin in different concentrations (75, 100, and 150 μM) for 3 h before exposure to 4 Gy x-ray with a dose of 1.96 Gy/min. Radiation therapy was done in Guilan Irradiation Center. The samples were kept in a humidified atmosphere containing 5% CO2. After 48 h incubation, 6 µg/mL cytochalasin B (Sigma) was added, and the cells were harvested for 72 h. The cells were fixed in 3:1 (v/v) methanol/acetic acid. Afterward, the cells were dropped onto cooled slides with a Pasteur pipette and air-dried. The slides were stained in 10% Giemsa (Merck), and the MNs were scored in 500 binucleated cells. They were observed by a light microscope using a 400x magnification based on the criteria summarized by Fenech (2000). 
3. Results
The measurement of micronucleated and binucleated cells and the total number of micronuclei in 500 binucleated cells showed the following results. As it is clear from the results, in non-radiation groups, a significant difference was observed between the treated groups with 75 and 100 μM tolmetin and the control group (P<0.05). In all treated groups with tolmetin that also received radiation, a significant increase in the number of micronuclei was observed, which was more noticeable at concentrations of 100 and 150 μM. At the same time, tolmetin at the studied concentrations did not change the NDI index. However, a significant difference was observed between the radiation group with the control group regarding the NDI index (P<0.05).
4. Discussion and Conclusion
Resistance of cancer cells to ionizing radiation is a challenge in the radiotherapy regimen. Cancer cells activate different signaling pathways that lead to resistance to radiation-induced cell death [8]. Therefore, the use of agents or compounds that can sensitize tumor cells to radiation and maximize the dose of radiation to the patient is important. It is desirable that these compounds have no side effects, no cytotoxicity, and be cheap and available to the public. This study aimed to evaluate the radiosensitizing effect of tolmetin in radiotherapy treatment on human colon cancer cell line HT-29. Final results demonstrated that tolmetin has a radiosensitizing effect on HT-29 colon cancer cells, which depends on the tolmetin concentration. In addition, tolmetin has no cytotoxic effect on this cell line.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Guilan University of Medical Sciences (code: IR.GUMS.REC.1398.490). Al ethical principles were considered in this article.

Funding
This study was supported by the Deputy for Research and Technology of Guilan University of Medical Sciences.

Authors' contributions
Conceptualization, editing, review, and supervision: Mona Haddad Zahmatkesh; Writing the article: Saeedeh Rahmatpour; Radiotherapy: Hamid Saeedi Saedi; Experimental test: Mona Haddad Zahmatkesh and Saeedeh Rahmatpour.

Conflicts of interest
The author declared no conflict of interest.

Acknowledgements
The authors would like to thank the Vice Chancellor for Research of Guilan University of Medical Sciences.


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