Repeated sub-optimal photodynamic treatments with pheophorbide a induce an epithelial mesenchymal transition in prostate cancer cells via nitric oxide

Highlights

• Pheophorbide a/PDT releases nitric oxide in a dose-dependent way in tumor cells.

• Pba/PDT, through NO, modulates the NF-κB/YY1/Snail/RKIP circuitry dysregulated in tumor.

• The NF-κB/YY1/Snail/RKIP circuitry is linked to epithelial–mesenchymal transition.

• A repeated low-dose Pba/PDT treatment induces cell proliferation and EMT.

• NO induced by a repeated low-dose Pba/PDT treatment reveals a cytoprotective role.

Abstract

Photodynamic therapy (PDT) is a clinically approved treatment that causes a selective cytotoxic effect in cancer cells. In addition to the production of singlet oxygen and reactive oxygen species, PDT can induce the release of nitric oxide (NO) by up-regulating nitric oxide synthases (NOS). Since non-optimal PDT often causes tumor recurrence, understanding the molecular pathways involved in the photoprocess is a challenging task for scientists. The present study has examined the response of the PC3 human metastatic prostate cancer cell line following repeated low-dose pheophorbide a treatments, mimicking non-optimal PDT treatment. The analysis was focused on the NF-kB/YY1/RKIP circuitry as it is (i) dysregulated in cancer cells, (ii) modulated by NO and (iii) correlated with the epithelial to mesenchymal transition (EMT). We hypothesized that a repeated treatment of non-optimal PDT induces low levels of NO that lead to cell growth and EMT via the regulation of the above circuitry. The expressions of gene products involved in the circuitry and in EMT were analyzed by western blot. The findings demonstrate the cytoprotective role of NO following non-optimal PDT treatments that was corroborated by the use of L-NAME, an inhibitor of NOS.

Introduction

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic treatment that causes a selective cytotoxic effect against malignant cells. The procedure consists of the administration of a photosensitizer (PS) followed by irradiation at a wavelength falling within a PS absorbance band. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature and induction of a local inflammatory reaction [1]. The success of this therapy depends on the type and dose of PS, the time between PS administration and light exposure, the light dose and the fluence rate [2]. Recent data suggest that PDT-mediating changes to the tumor microenvironment can modulate the responsiveness of the treatment. Preclinical investigations indicated that combining PDT with targeted therapies directed at attenuating the pro-survival actions of the tumor microenvironment can enhance the therapeutic potential of PDT [3].

Several reports support the role of nitric oxide (NO) in PDT, by considering both the endogenous level of NO in the tumor [4], [5], [6] and NO directly induced by the photoactivated photosensitizers [7], [8], [9]. NO is known to directly influence a number of biological processes involved in PDT-induced anti-tumor effects [10], [11].

The biphasic role of NO in PDT seems to depend on a number of factors in the microenvironment, including the responsiveness of the tumor type, the redox state of the reaction, as well as the final intracellular concentration and the duration of intracellular exposure to nitric oxide [12], [13], [14]. Data in the literature indicated that low NO levels (<50 nM) enhance the survival, proliferation, and growth of tumor cells, by protecting the cells from apoptosis, whereas high NO levels (>300 nM) exhibit cytotoxic effects, by promoting DNA damage, protein dysfunctions, gene mutations and tumor cell death. Together, these effects may contribute to tumor regression [15], [16].

Concerning a low level of NO induced by PDT, several hypotheses have been proposed in order to explain underlying mechanisms of inhibition of apoptosis induced by NO [17], [18], [19], [20], [21]. It has been reported that NO inhibits the activation of caspases directly by S-nitrosylation [22], [23], by a cyclic guanosine monophosphate (cGMP)-dependent mechanism or by activating protein kinase G (PKG) [24]. Recently, Girotti and co-workers demonstrated that the cytoprotective role of NO in PDT is also due to the suppression of pro-apoptotic JNK and p38 MAPK activations [8], [18]. We propose another important pathway involved in tumor progression that is susceptible to NO, namely, the dysregulation of the NF-kB/YY1/Snail/RKIP circuitry. In a murine amelanotic melanoma we have reported that, according to the dose of the pheophorbide a/PDT (Pba/PDT) added to the cells, and consequently by the NO level induced by PDT, there were either progression or arrest of tumor growth, through the modulation of this circuitry [9], [18].

A disagreement also exists on the role of NO in the regulation of steps that are involved in the metastatic process. Metastasis is initiated with the acquisition of invasive and migratory properties by the tumor cells: a process known as epithelial to mesenchymal transition (EMT). EMT is a target of several constitutively activated survival pathways including the NF-kB pathway [25], [26], [27]. NF-kB induces EMT via the regulation of the expression of multiple matrix proteases, adhesion molecules and both angiogenic and invasive factors [25], [28], [29], [30], [31], [32].

Considering that (i) low NO concentrations have been reported to induce tumor cell migration and invasion [12], [15]; (ii) PDT modulates the effect of NF-kB in a dose-dependent manner [18], [33] through NO induction [9], [18], (iii) NF-kB regulates downstream gene products such as YY1 [34], a hallmark for the initiation of EMT during development and cancer metastasis and (iv) YY1 is overexpressed in prostate cancer cells [35], we investigated if a repeated low-dose Pba/PDT treatment can stimulate cell progression and induce EMT in a highly metastatic human prostate cancer cell line through the modulation of the NF-kB/YY1/RKIP circuitry.