Sub-additive effects of photodynamic therapy combined with erlotinib for the treatment of epidermoid carcinoma: An in vitro study

https://doi.org/10.1016/j.pdpdt.2017.03.010Get rights and content

Highlights

Abstract

Background

Photodynamic therapy (PDT) is an antitumour treatment that employs the combination of a photosensitive compound, oxygen and visible light. To improve the antitumour activity of PDT, the present study used the strategy of combining PDT with erlotinib (ERL), a drug frequently used in the treatment of epidermoid carcinoma.

Methods

An MTT cell viability assay was used to evaluate the cytotoxicity of PDT combined with ERL on A431 epidermoid carcinoma cells in vitro. This study evaluated the cytotoxicity of the following treatments: red laser irradiation (660 nm) at different power densities (1.25–180 J/cm2), the photosensitizer methylene blue (MB) at concentrations of 0.39–100 μM, PDT (12.5 μM MB and laser power densities from 1.25 to 180 J/cm2), and PDT (12.5 μM MB and a laser density of 120 J/cm2) plus ERL (1 μM).

Results

The laser power densities that were tested showed no cytotoxicity in A431 cells. MB showed a dose-dependent cytotoxicity. In PDT, an increase in the dose of light resulted in an increase in the cytotoxicity of MB. In addition, there was a sub-additive effect between PDT and ERL compared to the effect of each therapy alone.

Conclusions

The sub-additive effect between PDT and ERL suggests that their combination may be an important strategy in the treatment of epidermoid carcinoma.

Introduction

Photodynamic therapy (PDT) is increasingly used to promote the regression and disappearance of tumours. PDT is a treatment that combines a photosensitive compound administered topically or systemically, molecular oxygen present in the reaction medium, and a source of light. After application, the photosensitizer penetrates and is fixed in tumour tissues, receives the irradiating light, and directs it to the site to be treated. The activated photosensitizers transfer their excess energy to the surrounding oxygen to form singlet oxygen (1O2) or free radicals, which cause irreversible damage to diseased cells and tissues [1], [2], [3], [4].

However, the clinical use of photosensitizing compounds has been hindered by significant side effects, including damage to normal tissues due to poor tissue selectivity, hydrophobicity and environmental degradation [5]. Dyes, such as methylene blue (MB), have been studied and used in the application of PDT and as a commercially available photosensitizer in chemical reactions. These applications have led to the testing of MB against various cell lines as a possible candidate for the PDT treatment of cancer. Regarding its physicochemical properties, MB is hydrophilic, which determines many aspects of its pharmacology and intracellular localization [6]. In this regard, MB is one of the main photosensitizing agents used in PDT due to its good tissue penetration and low cytotoxicity. The absorption band of MB varies between 600 and 900 nm, and good tissue penetration and maximum absorption occurs at 660 nm [5], [7], [8], [9].

PDT has the advantage of selectively destroying tumour tissue while sparing adjacent normal tissue, resulting in a functional preservation of structures [10]. The effectiveness of PDT depends on the concentration of the applied photosensitizer and the availability of oxygen [11].

Although favourable clinical results have been obtained through the use of PDT alone, its combination with antitumour drugs, such as erlotinib (ERL), has improved its effectiveness as an anticancer therapy [12]. ERL is a selective, reversible inhibitor of protein tyrosine kinase and has an established antitumour effect. Its specific mechanism of action is the inhibition of the epidermal growth factor receptor (EGFR) [13]. EGFR is overexpressed in many cell types, including human epidermoid carcinoma cell lines (A431) [14]. A431 cells are used as a model to study the effect of drugs on cancers that overexpress EGFR [15], [16], [17]. According to Edmonds et al. inhibiting EGFR leads to an increase in the cytotoxicity of PDT through a mechanism that involves increased cell death by apoptosis [18].

Based on this observation, we hypothesized that the combination of MB in PDT with ERL would increase the antitumour activity of PDT in A431 epidermoid carcinoma.

Therefore, the objective of this study was to evaluate the effects of the antitumour agent ERL in combination with PDT on tumour cells in vitro.

Section snippets

Materials

ERL was obtained from US Biological (Salem, MA, USA). Methylene blue was purchased from Merck (Darmstadt, Hesse, Germany). Foetal bovine serum (FBS), trypsin/ethylenediaminetetraacetic acid (EDTA) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma (St. Louis, MO, USA). Amphotericin B-streptomycin was obtained from Gibco (Grand Island, NY, USA). The reagent 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was obtained from Invitrogen (Carlsbad, CA, USA).

In vitro cytotoxicity of red laser irradiation (660 nm)

Fig. 1 shows the in vitro cytotoxicity of red laser irradiation at different energy densities in A431 epidermoid carcinoma cells.

The data in Fig. 1 indicate that the red laser used in this study showed no destructive potential, and these results were independent of the energy density. This finding is interesting from a clinical standpoint, since the irradiated area is typically not restricted to the tumour lesion.

There are several studies evaluating the cytotoxicity of lasers emitting light in

Conclusions

The present study showed that the use of a low-power red laser (660 nm) at energy densities between 1.25 and 180 J/cm2 showed no cytotoxicity in the A431 cell line. The dose-response study of methylene blue showed that the cytotoxicity is dependent on the photosensitizer concentration. There was a sub-additive effect between PDT and erlotinib, demonstrating that the combination of new therapeutic modalities, such as PDT with chemotherapeutic drugs, represents an important strategy for the

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

We are grateful for CAPES, CNPq, FAPEMIG (Process: BPD-00484-14), Pró-Reitoria de Pesquisa − UFMG (PRPq), INCT/Nanobiofar, and the CNPq Nanofar network for providing financial support for this investigation.

References (37)

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