Photodynamic therapeutic role of indocyanine green in tumor-associated inflammation in skin cancer

Summary

Background

Indocyanine green (ICG) is a promising water-soluble photosensitizer for photodynamic therapy (PDT) of tumors. It was reported to have promising phototoxic effect on different cell lines. This study aimed to evaluate the efficacy of ICG as an efficient PS agent for skin cancer induced in mice.

Methods

Skin squamous cell carcinoma was induced in female CD-1 mice by 7,12-dimethylbenzanthracene and 12-O-tetradecanoyl-phorbol-13-acetate followed by an ICG/PDT treatment. The laser irradiation for PDT was adjusted to cover the whole body of the mice to make sure that the treatment protocol will be delivered to multiple tumors.

Results

The treatment of skin cancer by ICG/PDT using intravenously injected ICG initiated tumor cell death and significantly decreased cell proliferation as indicated by the reduction in proliferating cell nuclear antigen positivity. A significant reduction in the inflammatory mediators; tumor necrosis factor-α, nitric oxide and 5-lipoxygenase was reported, however the level of cyclooxygenase-2 (COX-2) was significantly elevated after ICG/PDT treatment.

Conclusion

The proposed ICG/PDT treatment modality showed a significant anti-tumor and anti-inflammatory activity against skin cancer accompanied with COX-2 elevation.

Introduction

Photodynamic therapy (PDT) is increasingly applied as an alternative treatment for superficial cancer. PDT involves photosensitizer (PS) administration followed by an illumination of tumor with light of appropriate wavelength to activate the PS [1]. Activation of PS transforms its ground state (¹PS) into an excited singlet state (¹PS*). To obtain therapeutic effect, PS must undergo electron spin conversion to its triplet state (³PS*). In the presence of oxygen, the excited molecule can react with a biological substrate (membrane lipids, proteins, nucleic acids, etc.) and form free radicals, which can interact with ground-state molecular oxygen to produce reactive oxygen species (ROS) (type I reaction). Alternatively, the energy of the excited PS can be directly transferred to oxygen to form non-radical but highly reactive singlet oxygen (¹O₂), which is the most damaging species generated during PDT (type II reaction) [2]. The in situ generation of ¹O₂ can directly kill tumor cells by the induction of apoptosis and necrosis and can damage the tumor vasculature and the surrounding healthy vessels, resulting in indirect tumor death via the induction of hypoxia and starvation. In addition, PDT is able to initiate an immune response against the remaining tumor cells [3]. The efficacy of PDT in the treatment of cancer depends on the PS type, concentration, intracellular localization, light dose (fluence) and dose rate (fluence rate), oxygen availability, and the treatment regimen given [1].

Skin cancers are ideally suited for PDT. In an early clinical trial, complete response rates of 85% were achieved by the use of the PS hematoporphyrin derivative followed by red light [4]. Since then, several studies confirmed that PDT, using different sensitizers, achieved response rates for superficial skin cancers that are equivalent to those achieved by conventional methods (e.g. cryotherapy and surgical excision). Today three more sensitizers are approved for clinical use; 5-aminolevulinic acid (5-ALA, Levulan^®), the methyl ester of ALA (Metvix^®, Photocure ASA), and meso-tetra-hydroxyphenyl-chlorin (mTHPC, Foscan^®) and PDT is becoming an established treatment modality for localized cancers including non-melanoma skin cancer [5], [6], [7], [8].

Indocyanine green (ICG; 4, 5-benzoindotricarbocyanine; C₄₃H₄₇N₂NaO₆S₂), is a negatively charged water-soluble anionic dye that is commonly used as an angiographic agent. ICG has a strong absorption in the near IR (between 700 and 800 nm), which is an important photochemical property, because it is known that melanins do not significantly absorb in this area [9]. Additionally, near IR light is characterized by a deep penetration into tissue [10]. The fact that ICG absorbs in the near IR region of the spectrum together with the fact that melanins do not absorb in this wavelength area is essential and important for assessing ICG/PDT in skin cancer treatment. In a previous study, ICG was reported to have a promising cytotoxic effect (at concentration 100 μM) on human (Sk-Mel-188) and mouse (S91) melanoma cells, where the surviving fraction of the treated cells decreased more than 10-fold (at 100 J/cm² with the fluence rate of 200 mW/cm²) compared with non-treated cells [11]. In an early study by our group [12], ICG concentration of 150 μM was selected as a safe effective dose for the treatment of Sk-Mel-28 melanomas cells and the experiment also indicated that at this dose of ICG the laser exposure (wavelength; 788 nm, average power; 400 mW and density; 57 mW/cm²) for 30 min or less led to <20% cell death, while exposure for 60 min resulted in dramatic membrane damage in 71% of the cells, which means that cell viability is both ICG concentration and light dose dependent. Recently a study was performed by our group [13] studying the phototoxic and genotoxic effect of ICG and ICG-entrapped in polymeric nanoparticles on two different cell lines; human breast adenocarcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2) using different concentrations of ICG (12.5–200 μM), laser dose (wavelength; 807 nm, average power; 400 mW and power density; 51 mW/cm²), and 20 min time period of exposure. The results revealed that ICG has a good phototoxic effect and this phototoxic effect increased with increasing in the ICG concentration. ICG also showed a DNA damage effect at high concentration on tumor cells [13]. The present study is an in vivo approach to evaluate the efficacy of ICG as an efficient PS agent for skin cancer induced in two-stage murine skin carcinogenesis model.