Photodynamic action of methylene blue in osteosarcoma cells in vitro

https://doi.org/10.1016/j.pdpdt.2013.09.003Get rights and content

Summary

Background

Osteosarcoma is a common malignant bone tumor which threatens the life of young people worldwide. To explore alternative strategy for combating osteosarcoma, a light-emitting diode (LED) that activates methylene blue (MB) was used in the present study to investigate cell death of osteosarcoma-derived UMR106 cells.

Materials and methods

Photocytotoxicity in UMR106 cells was investigated 24 h after photodynamic activation of MB using sulforhodamine B (SRB) assay and light microscopy. Apoptosis induction was observed 24 h after photodynamic treatment using a confocal laser scanning microscopy (CLSM) with Hoechst 33342 staining. The change in mitochondrial membrane potential (MMP) was analyzed using a flow cytometry with rhodamine 123 staining.

Results

MB under red light irradiation caused a drug-concentration (0–100 μM) and light-dose (0–32 J/cm2) dependent cytotoxicity in UMR106 cells. The SRB assay and light microscopy observed a significant decrease in the number of UMR106 cells attached to the bottom of culture well after LED light-activated MB (100 μM, 32 J/cm2). Nuclear shrinkage, chromatin condensation and fragmentation were found in the treated cells by nuclear staining. In addition, flow cytometry showed that the MMP in UMR106 cells was rapidly reduced by photo-activated MB (100 μM, 32 J/cm2).

Conclusion

Photodynamic action of MB under LED irradiation could remarkably kill osteosarcoma cells and induce cell apoptosis as well as MMP collapse.

Introduction

Osteosarcoma is one of the commonest malignant bone tumors threatening the life of young people worldwide [1]. The survival of patients with osteosarcoma has been enhanced by advanced modern remedies [2], [3], however, the long-term efficacy of the current therapeutic modalities is very limited due to drug-resistance and cancer recurrence [4], [5]. Therefore, novel and more effective strategies are in urgent need for eradicating osteosarcoma cells.

Photodynamic therapy (PDT) is an alternative for combating malignant tumors [6], [7], [8] via photochemical action-induced cytotoxic reactive oxygen species (ROS) from photosensitizers retaining in over-growing tumor cells activated by appropriate light [9]. In PDT photosensitizer and light source are two key components. In order to explore photodynamic therapy as a new therapeutic technique, we have successfully set up new light sources using light-emitting diodes (LED). Our previous study showed that red light from this LED light source could effectively activate pyropheophorbide-α methyl ester (MPPa) and pheophorbide a (Pa) to kill various tumor cells in vitro [10], [11]. Blue light from this LED light source could remarkably activate hypocrellin B to deactivate ovarian and breast cancer cells [12], [13]. Methylene blue (MB) is a second-generation photosensitizer from phenothiazine dyes with remarkable photocytotoxicity of tumor cells. Emerging studies have shown that MB was retained in the malignant tissues [14]. Recently, Matsubara et al. reported that MB under the irradiation of light from a 500 W xenon lamp source had a strong photocytotoxic effect on mouse osteosarcoma (LM8) cells through affecting cytoplasmic ballooning, and inducing necrosis and apoptosis [15]. In this present study, we used red light from a new LED light source to activate MB for observing the killing efficacy of LED-activated MB on osteosarcoma-derived UMR106 cells in vitro and explore apoptotic cell death and mitochondrial membrane potential (MMP) of UMR106 cells induced by photodynamic action of MB.

Section snippets

Photosensitizer and light source

Methylene blue (MB) (C16H18ClN3S, M.W. 319.90, ≥82%) was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). A stock solution (3 M) was made in phosphate buffer saline (PBS) and stored in the dark at −20 °C until used.

The light source was from a red LED with the wavelength of 630 nm. Its highest power density is 106.5 mW/cm2 with a uniform distribution over an area of 78.5 cm2. The samples were exposed to the LED light at the energy density of 0, 2, 4, 8, 16 and 32 J/cm2, respectively.

Cell culture

Rat

Photocytotoxicity of MB in UMR 106 cells

The photocytotoxicity of UMR106 cells by LED-activated MB was measured using SRB assay and observed under a light microscopy. The cells were incubated in different MB concentrations (0–100 μM) for 1 h and exposed to red light from LED light source with various light energies (0–32 J/cm2). No significant dark cytotoxicity of MB was observed in the range of 0–100 μM. MB under the light irradiation caused a concentration-dependent cytotoxicity in UMR106 cells, and photocytotoxicity of MB in UMR106

Discussion

About 30% of osteosarcomas are found to be multidrug-resistant [16]. However, surgical resection is very difficult to fully remove osteosarcoma cells. The residual malignant cells are a main cause of osteosarcoma recurrence. Gong's study demonstrated that benzochloroporphyrin derivative-17-mediated PDT exhibited a cytotoxic effect on cultured LM-8 osteosarcoma cells, and the rate of local recurrence and the surgical margins were reduced by PDT after marginal resection of tumors [17]. Therefore,

Acknowledgements

This work was supported by the GRF grant from Hong Kong RGC (476912) and Innovation and Technology Fund of Shenzhen (CXZZ20120619150627260), and the Direct Grant from the Chinese University of Hong Kong (2030446, 4053026). We express our sincere thanks to Miss Winnie Lee for her helpful assistance.

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