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ORIGINAL ARTICLE
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Duramycin radiosensitization of MCA-RH 7777 hepatoma cells through the elevation of reactive oxygen species


1 Department of Medicine, Shanxi Medical University, Taiyuan, Shanxi, China; Department of Radiology, Northwestern University, Chicago, Illinois, USA
2 Department of Radiology, Northwestern University, Chicago, Illinois, USA
3 Department of Radiology, Northwestern University; Research Resource Center, University of Illinois at Chicago, Chicago, Illinois, USA

Correspondence Address:
Andrew Christian Larson,
Department of Radiology, Feinberg School of Medicine, Northwestern University, 737 N Michigan Avenue, 16th Floor, Chicago, Illinois 60611
USA
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_284_18

 > Abstract 


Objective: The objective of this study is to explore the radiosensitization effects of duramycin against the liver cancer hepatoma cells and relationship to reactive oxygen species (ROS) generation.
Materials and Methods: MCA-RH 7777 cells were treated with various combinations of duramycin concentrations and radiation doses. After the treatment, cell viabilities were determined by a cell proliferation assay; intracellular ROS levels were detected with the flow cytometric method.
Results: MCA-RH 7777 cell viability was found significantly reduced after combining duramycin and radiation exposure (comparing to that of either treatment alone). Increased intracellular ROS levels were observed in cells treated with combinations of duramycin and radiation.
Conclusion: Duramycin increased the intracellular ROS generation and also increased the radiosensitivity of MCA-RH 7777 cells.

Keywords: Duramycin, radiosensitivity, reactive oxygen species



How to cite this URL:
Yang B, Huang X, Li W, Mouli S, Lewandowski RJ, Larson AC. Duramycin radiosensitization of MCA-RH 7777 hepatoma cells through the elevation of reactive oxygen species. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269910




 > Introduction Top


One of the major hurdles in treating cancer is that tumor cells develop resistance to radiotherapy.[1] Improving radiosensitivity of cancer cells is an urgent clinical need with a goal of achieving curative effects while protecting the surrounding normal tissues. Mounting evidence has indicated that radioresistance of cancer cells can be reduced by anticancer agents that induce the production of intracellular reactive oxygen species (ROS).[2]

As a potential effective antitumor agent, duramycin was reported to specifically bind to phosphoethanolamine (PE) in the target cell membrane to induce calcium ion (Ca2+) release from cancer cells.[3],[4] Calcium signaling pathways can interact with cellular signaling systems such as ROS systems that play an important role in fine-tuning cellular signaling networks.[5] Thus, duramycin may sensitize cell to radiotherapy through the changes in cellular ROS level.

In the present study, we treated a hepatoma cell line MCA-RH 7777 with various concentrations of duramycin and different radiation levels to observe the cytotoxicity and radiosensitizing effects of duramycin.


 > Materials and Methods Top


Cell culture and agents

Hepatoma cell line, MCA-RH 7777, was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% fetal bovine serum (Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% CO2. Duramycin (Sigma-Aldrich) was dissolved in hydrogen chloride to a stock concentration of 10 mM and stored at 4°C.

Cell irradiation

MCA RH-7777 cells (4 × 105) were seeded into four 96-well plates. After incubation for 1 day, cells on each plate were divided into four groups and treated with duramycin of four different concentrations (0, 1, 2, and 4 μM) for 4 h, respectively; then, each plate was irradiated with γ-(60 Co) radiation with one of four different dose levels (0, 2, 4, and 6 Gy). Cell viability was measured at 48 h after irradiation.

Cell viability test with 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid assay

Cell viability was measured at 48 h after irradiation with 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid (MTT). The MTT assay was measured as previously described.[6] Briefly, cells in each well were incubated with 100 μL of MTT solution (5 mg/mL) for 4 h at 37°C. MTT solution was then discarded and 50-μL dimethyl sulfoxide was added to dissolve insoluble formazan crystal, and the plates were incubated at 37°C for 30 min. Optical density (OD) of the solutions was measured at 490 nm using a spectrophotometer reader (BIO-TEK® ELx800™, USA). Cell viability was calculated as where A and B represent the OD of treated and untreated cells, respectively.

Reactive oxygen species levels examined by flow cytometry

The intracellular ROS level was determined by flow cytometry analysis according to the previously described methods.[7] After treatment with duramycin and irradiation, cells were resuspended in phosphate-buffered saline (PBS) containing 3 μM of 2',7'-dichlorofluorescein-diacetate (CM-H2DCFDA) (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 30 min. Subsequently, the cells were harvested, rinsed, and resuspended in the PBS solution. The intracellular ROS level was determined by fluorescence-activated cell sorter analysis.

Statistical analysis

The experimental data were expressed as mean ± standard error of the mean. Analysis of variance (ANOVA) was used to compare the cell viability from each of the four duramycin concentrations and radiation doses. Post hoc testing was performed using the Tukey method. All the statistical analyses were performed using SPSS version 18.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.


 > Results Top


Representative colony plates of MCA-RH 7777 cells after treatment with duramycin and radiation are shown in [Figure 1]. The combination of duramycin and radiation (lower panel) demonstrated significant inhibition of cell clonogenicity comparing to solely duramycin treatment (upper panel). After treatment for 48 h, cell viabilities of MCA-RH 7777 cells treated with 0-, 1-, 2-, and 4-μM duramycin were 1 ± 0.07, 0.75 ± 0.03, 0.63 ± 0.02, and 0.50 ± 0.06, respectively. However, as shown in [Figure 2], ANOVA analysis indicated significantly lower viability (P< 0.05) after combinations of each level of radiation and duramycin comparing to that of duramycin treatment alone. No significant differences of cell viability were found between radiation doses at 0-, 1-, and 4-μM duramycin. Cell viability was found decreased with increase in duramycin dose.
Figure 1: Cell viability treated with duramycin and radiation. Upper panel (a-d) treated with solely duramycin; lower panel (e-h) treated with combinations of 6 Gy of radiation and duramycin concentrations of 0, 1, 2, and 4 μM, respectively. Scale bar: 2 mm

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Figure 2: Effect of duramycin and radiation on the cell viability of MCA-RH 7777 at 48 h of following treatment. Cells were treated with four concentrations of duramycin for 4 h and then irradiated with four dose levels

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ROS was evaluated by flow cytometry using H2DCFDA. Representative flow cytometric histograms show the base line fluorescence level determined by the control cells without duramycin and radiation treatment [Figure 3]a and fluorescence level of the MCA-RH 7777 cells treated with both duramycin and radiation [Figure 3]b that indicated a significant increase in ROS generation. As shown in [Figure 3]c, for cells treated solely with duramycin, intracellular ROS levels increased as the concentration of duramycin increased. However, for cells treated with a combination of duramycin and with each applied radiation dose from 2 to 6 Gy, the maximum intracellular ROS level in the MCA-RH 7777 cells was found for duramycin exposure concentration of 2 μM. At duramycin concentrations of 1, 2, and 4 μM, intracellular ROS levels were significantly higher for cells irradiated at the radiation doses of 2, 4, and 6 Gy (P< 0.05) when comparing to the corresponding control cells with radiation treatment alone.
Figure 3: Intracellular reactive oxygen species production analyzed by flow cytometry analysis of MCA-RH 7777 cells treated with duramycin and radiation. Representative flow cytometric histograms of control (a) and cells treated with duramycin and radiation (b). Group average of fluorescence intensities indicating the measured intracellular ROS levels for four radiation doses at four duramycin concentrations (c). Same florescence threshold applied to all the cells

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 > Discussion Top


Radiation tolerance is one of the top challenges in cancer therapy. As a dominant form of cancer treatment, concurrent administration of radiation and chemotherapy relies on radiosensitization process of chemotherapeutic agents. In our study, we found a combination of duramycin with radiation-elevated radiosensitization of MCA-RH 7777 cells, and the increase of radiosensitivity was related to intracellular ROS accumulations induced by duramycin. Our finding could provide new ideas for the treatment of liver hepatoma and other cancers.

It was widely accepted that radiotherapy and many chemotherapy agents act by producing free radicals.[8] Free radicals and ROS attack the covalent bonds of the DNA, resulting in cell apoptosis.[9] DNA damage inflicted by radiation can be enhanced by elevating cellular ROS levels. Our results showed that, except for the duramycin concentration of 4 μM, duramycin-treated MCA-RH 7777 cells had significantly higher ROS level comparing to the controls [Figure 3]c, indicating that duramycin might enhance the efficacy of radiation against MCA cells due to this ROS generation. For cells treated with 4-μM duramycin, while highest ROS level was observed in samples treated with duramycin alone, significantly lower ROS levels (P< 0.05) were found in cells treated with combinations of duramycin and radiation [Figure 3]c. This phenomenon can likely be explained given that the ROS measurements are impacted by altered cell viabilities at these dose levels. Otherwise, our results demonstrated a trend that cellular ROS levels changed in a concentration/dose-dependent manner, which correlated well with cell death levels as analyzed by MTT methods [Figure 2]. Further in vivo studies are now necessary to determine the optimized combination of duramycin concentration and radiation dose.

The specific mechanisms of interaction between radiation and duramycin are still unclear. The previous study demonstrated that99m Tc-duramycin specifically accumulates in apoptotic tumors.[10],[11] The high accumulation of duramycin in tumors can lead to increased cellular ROS levels in cancer cells through PE binding and the Ca2+ release.[4],[11] Increased ROS levels can enhance DNA damage to the cancer cells. Thus, duramycin can essentially sensitize radiotherapy of cancers through intracellular ROS changes. However, the potential of combining duramycin with radiation as a novel effective method for cancer therapy still needs to be further investigated to better understand the mechanism by which duramycin improves the effectiveness of radiation therapy, both in model systems and in patients.


 > Conclusion Top


Our study demonstrated that duramycin enhanced the radiosensitivity of MCA-RH 7777 hepatoma cells. This observed radiosensitization process may relate to the ROS changes induced by the specific binding of duramycin within the exposed cancer cells.

Financial support and sponsorship

This study was supported by NIH NCI RO1CA181658.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Willers H, Held KD. Introduction to clinical radiation biology. Hematol Oncol Clin North Am 2006;20:1-24.  Back to cited text no. 1
    
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Tong L, Chuang CC, Wu S, Zuo L. Reactive oxygen species in redox cancer therapy. Cancer Lett 2015;367:18-25.   Back to cited text no. 2
    
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Yates KR, Welsh J, Udegbunam NO, Greenman J, Maraveyas A, Madden LA, et al. Duramycin exhibits antiproliferative properties and induces apoptosis in tumour cells. Blood Coagul Fibrinolysis 2012;23:396-401.  Back to cited text no. 3
    
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Broughton LJ, Crow C, Maraveyas A, Madden LA. Duramycin-induced calcium release in cancer cells. Anticancer Drugs 2016;27:173-82.  Back to cited text no. 4
    
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Görlach A, Bertram K, Hudecova S, Krizanova O. Calcium and ROS: A mutual interplay. Redox Biol 2015;6:260-71.  Back to cited text no. 5
    
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Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.  Back to cited text no. 6
    
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Ameziane-El-Hassani R, Boufraqech M, Lagente-Chevallier O, Weyemi U, Talbot M, Métivier D, et al. Role of H2O2 in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells. Cancer Res 2010;70:4123-32.  Back to cited text no. 7
    
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Dayal R, Singh A, Pandey A, Mishra KP. Reactive oxygen species as mediator of tumor radiosensitivity. J Cancer Res Ther 2014;10:811-8.  Back to cited text no. 8
    
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Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J, et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44-84.  Back to cited text no. 9
    
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Elvas F, Boddaert J, Vangestel C, Pak K, Gray B, Kumar-Singh S, et al. 99mTc-duramycin SPECT imaging of early tumor response to targeted therapy: A comparison with 18F-FDG PET. J Nucl Med 2017;58:665-70.  Back to cited text no. 10
    
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Broughton LJ, Giuntini F, Savoie H, Bryden F, Boyle RW, Maraveyas A, et al. Duramycin-porphyrin conjugates for targeting of tumour cells using photodynamic therapy. J Photochem Photobiol B 2016;163:374-84.  Back to cited text no. 11
    


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  [Figure 1], [Figure 2], [Figure 3]



 

 
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