Photochemotherapeutic effects of UV-C on acridine orange in human breast cancer cells: potential application in anticancer therapy

Abstract

The photochemical effects of Ultra Violet-C (UV-C) radiation on the fluorochrome, acridine orange (AO) in human breast cancer cells (MCF-7) has been investigated for effective anticancer therapy. Fluorescence enhancement of AO under UV-C irradiation has been evaluated in a cell free environment using fluorescent spectral studies. Further, its photo-toxic effects were assessed using MTT assay, NBT assay and apoptotic studies through Hoechst staining and fluorescent activated cell cycle analysis. The results demonstrate that AO under UV-C exhibits enhanced fluorescence and dose dependent cytotoxicity with significant ROS production at higher doses compared to light and dark toxicity. Apoptotic studies reveal that higher exposure to UV-C of intracellular AO significantly increases the Sub-G1 fraction and morphological changes were distinct compared to the control cells. These results demonstrate that UV-C could be an ideal light delivery system for AO mediated photo-chemotherapy and can be utilized for effective anti-cancer therapy.

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Introduction

Targeted drug delivery and drug resistance mechanism limit the efficacy of chemotherapeutic drugs against cancer cells.¹ Photodynamic and radiotherapies are the two effective alternative strategies trialed in recent years, apart from chemotherapy in cancer research.^2,3 Radiotherapy involves the administration of high energy radiation (X-rays, γ-rays, UV rays, etc.) to destroy cancer cells. Intensity modulated radiation therapy and image-guided radiation therapy show advanced insights into radiation incurred cancer treatment.⁴ Photodynamic therapy involves administration of a photosensitizer (PS) into the cancer cells followed by the exposure of appropriate light, which triggers the cells to undergo oxidative stress leading to apoptosis and necrosis of cancer cells. The effective photodynamic therapy depends upon the selection of an appropriate photosensitizer and its localization, nature of light source and targeted cell types.^5–8

Photo-chemotherapy (PCT) involves administration of ultraviolet (UV) radiation and pharma active molecules for enhanced cellular reactivity.^9,10 Commonly UV-A and B radiations are used in PCT to sensitize pharma active molecules or cells, for topical cancer treatments. Ultra violet C, a shortwave radiation (λ = 280–100 nm) shows effective photochemical response to biomolecules including nucleic acids. Purines and pyrimidine's absorb UV-C rapidly and induce double strand breaks in DNA molecules.¹¹ Although UV-C is completely absorbed by the earth atmosphere, its effect on DNA damage mediated apoptosis has been extensively studied.¹² In addition UV-C has been widely used in mutation studies and shows higher sensitivity to PDT resistant cells.^13,14 Recent results indicate that UVC has direct effect on melanoma growth as well as an anti-angiogenesis effect¹⁵ and it also enhances the photodynamic therapy in combination with fluorescent proteins against cancer cells.¹⁶ However, the effectiveness of a UV-C radiation relies on its dosage form rather than its nature.

Acridine orange (AO), a non-toxic nucleic acid binding photosensitizer has recently been used in the field of photodynamic therapy due to its multifunctional properties like cellular permeability,¹⁷ tumor specific accumulation,¹⁸ fluorovisualization effects¹⁹ etc. It is highly permeable and can be easily localized in the cells, due to its low molecular weight and hydrophobic nature. Being a weak basic dye it can bind easily with acidic structures such as DNA, RNA and lysosomes by intercalation and electrostatic attraction.^20,21 It also serves as a fluorescent pH indicator, when AO binds with nucleic acids it appears orange in color at pH 8 and green at pH 10. AO, shows strong photodynamic activity against wide range of tumor cells and shows stronger cytotoxic effects under blue light illumination and also at low doses of X-ray radiation.¹⁷

In the present study the photochemical effects of UV-C was assessed using the water soluble fluorescent photosensitizer, AO in human breast cancer cell line, MCF-7. Fluorescent studies reveals that molecular changes of acridine orange under UV-C irradiation shows enhanced fluorescent intensities with respect to exposure time of UV-C. This enhanced fluorescent intensity of AO and its potential photo-toxicity results in apoptosis in MCF-7 cells. This fluorescent enhanced effect of AO under UV-C with apoptotic induction could provide an insightful in the area of cancer research.

Experimental

Cell lines and maintenance

Human breast cancer cell line MCF-7 was obtained from National Centre for Cell Sciences (NCCS, India) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin–streptomycin (100 μg mL⁻¹) at 37 °C in 5% CO₂ environment.

Light source

For irradiation of cells, a 30 W UV lamp (Philips) emitting UV-C with a wavelength of 280 nm at a fixed distance of 15 cm was used for irradiation of cells and cell free AO during light treatments. The total fluence rate reaching the samples was maintained at 0.5 W cm⁻² using power meter (PM100D, Thor labs).

Photo-chemotherapy (PCT) treatment

The experimental setup for the UV-C mediated PCT includes six treatment groups as follows: acridine orange alone (AO, 1 μg mL⁻¹), UV-C alone (10 min exposure), PCT-1 (1 μg mL⁻¹ AO + 1 min UV-C), PCT-5 (1 μg mL⁻¹ AO + 5 min UV-C), PCT-10 (1 μg mL⁻¹ AO + 10 min UV-C) and control cells (without any treatment). All photo treatments were carried out in dark conditions and the cells were exposed to UV-C (1, 5 and 10 min) by direct contact of culture wells without lid covers in sterile chamber. Control cells were maintained without any drug and light treatments.

Fluorescent spectral characteristics of AO under UV-C

The influence of UV-C on fluorescent spectra of AO was analyzed using spectrofluorometer. The fluorescence emission spectra were recorded using a spectrofluorimeter (Horiba Fluromax4) at an excitation and emission wavelength of 490 nm and 520 nm. The fluorescent intensity of pure AO (1 μg mL⁻¹) and different levels of UV-C exposed AO (1, 5 and 10 min) were analyzed in a cell free environment.

In vitro cytotoxicity

The in vitro photo-toxicity of the UV-C in the presence and absence of AO in MCF-7 cells were assessed using MTT assay. In brief, cells were grown in DMEM medium, seeded at a density of 5 × 10⁴ cells per well in a 96-well tissue culture plate. After 24 h, the medium was replaced with a fresh medium consisting of different concentrations of AO (0.5, 1, 1.5, 2, 2.5 and 3 μg mL⁻¹) and incubated for 30 min. After incubation, AO treated cells were exposed to UV-C under different times of exposure (1, 5 and 10 min). Control cells were also maintained without AO and/or photo treatments. After the experimental period, the cells were incubated for 30 min and the medium was removed and the cells were treated with 10 μL of MTT dye (5 mg mL⁻¹) and further incubated for 4 h. After incubation, the formazan crystals were dissolved by adding 100 μL of DMSO and the absorbance was read at 590 nm using a multi-well plate reader (Bio-Rad, USA).

Morphological analysis of PCT

The effect of UV-C on cellular morphology of MCF-7 in the presence or absence of AO during PCT was determined using phase contrast microscopy (Nikon Eclipse TS100) and fluorescent microscopy (Nikon Eclipse 80i). Cells were grown on cover slips and incubated for 24 h. The confluent cells were treated with AO (1 μg mL⁻¹) and incubated for additional 30 min. After incubation, AO treated cells were exposed to UV-C under different times of exposure (1, 5 and 10 min) and incubated for 30 min. Control cells were maintained without any AO and UV-C treatments. After incubation time, cells were washed twice with ice-cold PBS (pH 7.4) and fixed on a clean glass slide by inversion and were directly observed under phase contrast and fluorescent microscope using green filter.

ROS determination by NBT reduction method

The effect of PCT on the production of superoxide anion (O₂⁻) in MCF-7 cells was measured using NBT method. Briefly cells were cultured on cover slips and photo treatments were carried out as described above. After the PCT treatment, the medium was completely removed and 0.1% NBT dye (100 μL) was added to each well and incubated in dark for 30 min. After incubation, NBT was removed and the wells were washed twice with PBS and methanol. The NBT deposited in the cells were then dissolved by adding 120 μL of 2 M KOH and 140 μL of DMSO with gentle shaking. The blue colored dissolved NBT solution was then transferred to fresh 96-well plate and read absorbance at 620 nm using micro plate reader (i-Mark, BIO-RAD).

Hoechst 33258 staining

The chromatin condensation and nuclear changes during early apoptosis induced by PCT of UV-C on AO was analysed by Hoechst 33258 staining method. In brief, PCT treated MCF-7 cells from 24-well plates were trypsinised, washed twice with PBS (pH = 7.4) and 20 μL of the cell suspension was mixed with equivalent volume of Hoechst 33258 and incubated for 10 min in dark. The nuclear condensation and apoptotic changes of the treated cells were visualised under fluorescent microscope using blue filter (Nikon Eclipse 80i).

FACS analysis

For cell cycle analysis, MCF-7 cells were grown on 6 well plates and were treated with AO (1 μg mL⁻¹), incubated for 30 min and subject to UV-C treatment at different times of exposure (1, 5 and 10 min). Control cells were maintained without AO/UV-C treatment. Cells were harvested, centrifuged (1000 rpm, 3 min), washed with PBS and fixed in 3 mL ice-cold 70% ethanol. After overnight incubation cells were centrifuged and resuspended with Propidium Iodide (10 μg mL⁻¹) and RNase A (100 μg mL⁻¹) at 37 °C for 30 min. The cells were then subjected to FACS Calibur instrument (BD Biosciences, San Jose, CA) equipped with Cell Quest 3.3 software.

Statistical analysis

All experiments were done in triplicate and the results were expressed as mean ± standard deviation. The experiments were analyzed by one way and two way ANOVA using GraphPad Prism 4.0 (GraphPad Software, San Diego; CA; USA). P < 0.05 was considered statistically significant.

Results and discussion

Photochemical effects of UV-C on AO

In the present study the photochemical effects of UV-C was assessed using the water soluble fluorescent photosensitizer, AO. The chemical stability of the AO before and after UV-C radiation was assessed by monitoring its optical property (absorption and fluorescence). As a light sensitive molecule, AO shows λ_max at 490 nm and emission wavelength at 520 nm (Fig. 1). At the fixed concentration of AO (1 μg mL⁻¹), the fluorescent spectrum was recorded before and after UV-C exposure. The potential impact due to photochemical effects of UV-C on AO was assessed by varying the time of irradiation (1, 5 & 10 min). Interestingly, the result shows that the fluorescent intensity of AO increases with higher UV-C exposure in a dose dependent manner (Fig. 2). UV-C exposure for a period of 10 min shows three-fold increase in fluorescent intensity of AO as compared to its initial fluorescence, suggesting potential influence of UV-C radiation on AO. The observed enhancement in the fluorescence of AO due to UV-C radiation could possibly be due to the molecular changes like chemical bond breakage and photon energy absorption. Though the term PCT is most commonly used UV-A treatment, our results demonstrate that it's not limited to UV-A. Compared to UV-A and UV-B radiation, UV-C has short penetrating power and does not permeate into solid bodies. These properties are essentially suitable for topical photodynamic therapy against cancer cells.^22–24 Apart from the wavelength of light, luminescence of the light is also an essential factor for the effective photodynamic therapy.

Fig. 1 (A) Absorption and fluorescent spectrum of a photosensitizer acridine orange, (B) chemical structure of AO.

Fig. 2 Spectrofluormetric analysis of enhanced fluorescent intensity of AO under UV-C exposure. Insert shows the peak intensity of AO under different time of UV-C exposure.

Intracellular distribution of AO under UV-C irradiation

In order to understand the impact of UV-C and AO mediated PCT in cells, we extended the study to in vitro cell culture model. AO being a hydrophobic cationic dye, it is highly permeable and stains both live and dead cells. Furthermore, the cellular uptake and intracellular distribution of AO before and after UV-C exposure in MCF-7 cells was monitored using fluorescent microscopy. AO shows higher fluorescence intensity with respective time of exposure to UV-C (10 min) and shows uniform distribution in the cytoplasm (Fig. 3). This fluoro-visualisation effect of AO under UV-C illumination shows enhanced fluorescence in a dose dependent manner as previously illustrated in a cell-free environment. The acidic environment in tumor, retains AO more efficiently compared to the normal tissue.¹⁸

Fig. 3 Cellular uptake and intra cellular distribution of AO during PCT in MCF-7 cells. Scale bar = 0.1 μm.

In vitro photo-toxicity of AO under UV-C irradiation

Influence of light on the AO employing in vitro cell culture model has been studied. The in vitro PCT effects of the metachromatic fluorescent photosensitizer AO under the influence of UV-C radiation was investigated in human breast cancer cell MCF-7. The in vitro dark cytotoxicity and photo-toxicity of AO with and without UV-C exposure was assessed by altering the AO concentration (0.5, 1, 1.5, 2, 2.5 and 3 μg mL⁻¹) as well as the UV-C exposure time (0, 1, 5 and 10 min). Control cells were maintained without any AO and UV-C treatment. Fig. 4 shows the photo-toxicity of AO with and without UV-C exposure. The percentage of cytotoxicity was significantly increased in UV-C exposed AO at different concentration as well as different time exposure. Particularly at AO dose of 1 and 1.5 μg mL⁻¹, the cell cytotoxicity increased up to 54% and 55% for 5 min UV-C exposure. Interestingly, no significant cytotoxicity was observed in either AO (27%) or UV-C (20%) alone treated cells even at higher concentration (3 μg mL⁻¹) and at higher exposure time (10 min) (Fig. 4 and 5). The results clearly demonstrate that the photo-toxicity of the AO has been greatly influenced by the UV-C irradiation.

Fig. 4 In vitro photo-toxicity of AO in MCF-7 cells with and without UV-C exposure. *** P < 0.001, ** P < 0.01, * P < 0.05 compared to AO control.

Fig. 5 In vitro photo-toxicity of Ultra violet-C rays on human breast cancer cell line, MCF-7.

Morphological analysis of MCF-7 cells

The extent of UV-C influenced photo-toxicity of AO on the morphology of human breast cancer cells (MCF-7) was also assessed using Phase contrast microscope (Nikon TSF-100, Japan). Fig. 6 shows the morphological assessment of MCF-7 cells before and after PCT. Control cells were maintained without any treatment protocols. At a fixed concentration of AO (1 μg mL⁻¹), UV-C shows enhanced cytotoxic potential with respect to different exposure time, this was further confirmed by the changes in the cell morphology. At 10 min of UV-C exposure, most of the cells swollen and died with ruptured cell membrane.

Fig. 6 Morphological assessment of MCF-7 cells before and after photo chemotherapy. (A) control cells were neither irradiated nor incubated with AO, (B) AO control(1 μg mL⁻¹), (C) UV-C (1 min) (D) UV-C (5 min), (E) UV-C (10 min), (F) PCT-1, (G) PCT-5, (H) PCT-10 min exposure. Scale bar = 10 μm.

Intracellular ROS production during PCT

Production of a ROS during light irradiation is a hallmark of a photodynamic therapy as well as photo radiation therapy.^25–28 The photo-toxicity of the AO under UV-C was assessed by superoxide anion production. ROS were produced upon UV-C irradiation on AO, due to the excitation of AO leading to the electron transfer reaction resulting superoxide anion production. AO-PCT treatment shows elevated levels of superoxide anion production compared to AO or UV-C alone treated cells as evidenced by NBT assay. Photo chemotherapy treatment shows significant superoxide anion production compared to control cells and AO alone treated cells. UV-C treatment upon higher exposure time (10 min), shows significant superoxide anion production (Fig. 7), our results corroborated with that of the other results.^25,26

Fig. 7 Intracellular superoxide anion production during PCT as determined by NBT reduction assay. *** P < 0.001, **P < 0.01 compared to control cells.

Apoptotic studies during PCT

To further confirm the potential of PCT using UV-C and AO and its impact on DNA, apoptosis and cell cycle, Hoechst staining and cell cycle analysis were carried out. Chromatin condensation and nuclear changes were observed enormously in PCT-treated cells than in control cells (Fig. 8). The apoptotic induction during PCT on MCF-7 cells was further assessed using percentage of cell cycle arrest (Sub G1 population).^28,29 Fig. 9A shows the DNA histogram of the MCF-7 cells subjected to PCT. The percentage of SubG1 level in control cells was 4.42 ± 0.43, whereas in AO alone or UV-C alone (10 min) treated cells shows 5.04 ± 0.68 and 6.02 ± 0.33 respectively. Interestingly, the SubG1 levels of PCT treated cells shows dose dependent arrest and distribution confirming the induction of apoptosis during PCT. Compared to control cells PCT-5 and PCT-10 shows significant increase in apoptotic cells (19.95 ± 8.19 and 24.95 ± 5.43). The results confirmed the photochemical effect of UV-C on AO and its ability to induce apoptosis in MCF-7 cells.

Fig. 8 Apoptotic induction of UV-C on AO in human breast cancer cell MCF-7 as evident by Hoechst staining. (A): control cells without AO and UV-C treatment, (B): AO control, (C): UV-C (10 min), (D): PCT-1, (E): PCT-5, (F): PCT-10. Scale bar = 1 μm.

Fig. 9 Apoptosis induction of AO during PCT using FACS analysis. (A–F) DNA histogram of MCF-7 cells under photodynamic treatment. (G) Sub G1 peak distribution indicating percentage of apoptosis.

Conclusions

In this study, we have demonstrated the photochemical effects of a UV-C radiation on AO and validated its potential application in PCT against human breast cancer cells. The extent of UV-C mediated AO induced photo-toxicity was assessed in MCF-7 cells. Fluorescent analysis reveals the molecular changes in AO when exposed to UV-C irradiation results in enhanced fluorescence in relation to the time of exposure. Higher photochemical reactivity of UV-C on AO was recorded in a cell free environment and its potential photo-toxicity in in vitro was assessed employing toxicology parameters using human breast cancer cells (MCF-7). The influence of UV-C on AO during PCT results in programmed cell death of MCF-7 cells, which has been confirmed by FACS analysis. Even at lower concentration, AO shows effective apoptotic efficiency in MCF-7 cells under UV-C exposure. These findings clearly demonstrated that the UV-C radiation could be an ideal source of light delivery system for PCT and can be utilized for effective anti-cancer therapy. Preclinical studies employing a mouse model for skin cancer are currently in progress in our laboratory to assess the in vivo efficacy of UV-C mediated PCT.

Acknowledgements

We gratefully acknowledge financial support from the Department of Biotechnology (Grant no. BT/PR12864/NNT/28/442/2009) and Department of Science & Technology (Grant no. SR/FT/LS-004/2009), Government of India, Ministry of Science and Technology, New Delhi, India. The authors are grateful to Dr M. V. Rao for fluorescent studies and Ms P. Prathiba and Mr S. Rajiv Gandhi for experimental support.

Notes and references

1. V. Rajiu, P. Arunkumar, H. Kalpana, K. B. Preetham, K. Jeganathan and K. Premkumar, J. Mater. Chem. B, 2013, 1, 1010–1018.
2. J. H. Lee, J. H. Song, S. N. Lee, J. H. Kang, M. S. Kim, D. I. Sun and Y. S. Kim, Cancer Res. Treat., 2013, 45, 31–39.
3. K. Plaetzer, B. Krammer, J. Berlanda, F. Berr and T. Kiesslich, Lasers Med. Sci., 2009, 24, 259–268.
4. T. S. Shylasree, A. Bryant and R. Athavale, Cochrane Database Syst. Rev., 2013, 28, 2.
5. D. E. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer, 2003, 3, 380–387.
6. F. F. Sperandio, S. K. Sharma, M. Wang, S. Jeon, Y. Y. Huang, T. Dai, S. Nayka, S. C. de Sousa, L. Y. Chiang and M. R. Hamblin, Nanomedicine, 2013, 9, 570–579.
7. P. Agostinis, E. Buytaert, H. Breyssens and N. Hendrickx, Photochem. Photobiol. Sci., 2004, 3, 721–729.
8. Y. Chen, M. Sajjad, Y. Wang, C. Batt, H. A. Nabi and R. K. Pandey, ACS Med. Chem. Lett., 2011, 2, 136–141.
9. A. Tanew, S. Radakovic-Fijan, M. Schemper and H. Hönigsmann, Arch. Dermatol., 1999, 135, 519–524.
10. K. Wolff, Br. J. Dermatol., 1990, 122, 117–125.
11. D. Kulms and T. Schwarz, Photodermatol., Photoimmunol. Photomed., 2000, 16, 195–201.
12. R. Takasawa, H. Nakamura, T. Mori and S. Tanuma, Apoptosis, 2005, 10, 1121–1130.
13. D. E. Godar, S. A. Miller and D. P. Thomas, Cell Death Differ., 1994, 1, 59–66.
14. H. Hama-Inaba, K. H. Choi, B. Wang, K. Haginoya, T. Yamada, I. Hayata and H. Ohyama, J. Radiat. Res., 2001, 42, 201–215.
15. M. H. Tsai, R. Aki, Y. Amoh, R. M. Hoffman, K. Katsuoka, H. Kimura, C. Lee and C. H. Chang, Anticancer Res., 2010, 30(9), 3291–3294.
16. M. Momiyama, A. Suetsugu, H. Kimura, H. Kishimoto, R. Aki, A. Yamada, H. Sakurada, T. Chishima, M. Bouvet, N. N. Bulgakova, I. Endo and R. M. Hoffman, Anticancer Res., 2012, 32(10), 4327.
17. K. Kusuzaki, H. Murata, T. Matsubara, H. Satonaka, T. Wakabayashi, A. Matsumine and A. Uchida, In Vivo, 2007, 21, 205–214.
18. K. Kusuzaki, H. Murata, T. Matsubara, S. Miyazaki, A. Okamura, M. Seto, A. Matsumine, H. Hosoi, T. Sugimoto and A. Uchida, Anticancer Res., 2005, 25, 1225–1235.
19. S. Ishikawa, R. Nemoto, S. Kanoh, K. Kobayashi and S. Ishizaka, Tohoku J. Exp. Med., 1984, 144, 265–271.
20. Y. Benchabane, C. Di Giorgio, G. Boyer, A. S. Sabatier, D. Allegro, V. Peyrot and M. De Méo, Eur. J. Med. Chem., 2009, 44, 2459–2467.
21. S. H. Tomson, E. A. Emmett and S. H. Fox, Cancer Res., 1974, 34, 3124–3127.
22. E. Ozcan, Z. Gok and E. Yel, Environ. Technol., 2012, 33, 1913–1925.
23. M. H. Tsai, R. Aki, Y. Amoh, R. M. Hoffman, K. Katsuoka, H. Kimura, C. Lee and C. H. Chang, Anticancer Res., 2010, 30, 3291–3294.
24. T. Yamauchi, S. Adachi, I. Yasuda, M. Nakashima, J. Kawaguchi, T. Yoshioka, Y. Hirose, O. Kozawa and H. Moriwaki, Int. J. Oncol., 2011, 39, 1375–1380.
25. H. Zhao, D. Xing and Q. Chen, Eur. J. Cancer, 2011, 47, 2750–2761.
26. H. Kolarova, P. Nevrelova, K. Tomankova, P. Kolar, R. Bajgar and J. Mosinger, Gen. Physiol. Biophys., 2008, 27, 101–105.
27. C. B. Oberdanner, K. Plaetzer, T. Kiesslich and B. Krammer, Photochem. Photobiol., 2005, 81, 609–613.
28. M. Kajstura, H. D. Halicka, J. Pryjma and Z. Darzynkiewicz, Cytometry, Part A, 2007, 71, 125–131.
29. H. M. Wang, C. C. Chiu, P. F. Wu, C. Y. Chen and J. Agri, J. Agric. Food Chem., 2011, 59, 8187–8192.

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