Photodynamic Therapy for X-ray-Induced Radiation-Resistant Cancer Cells

Abstract

Radiotherapy, in which X-rays are commonly used, is one of the most effective procedures for treating cancer. However, some cancer cells become resistant to radiation therapy, leading to poor prognosis. Therefore, a new therapeutic method is required to prevent cancer cells from acquiring radiation resistance. Photodynamic therapy (PDT) is a cancer treatment that uses photosensitizers, such as porphyrin compounds, and low-powered laser irradiation. We previously reported that reactive oxygen species (ROS) derived from mitochondria induce the expression of a porphyrin transporter (HCP1) and that laser irradiation enhances the cytotoxic effect. In addition, X-ray irradiation induces the production of mitochondrial ROS. Therefore, radioresistant cancer cells established with continuous X-ray irradiation would also overexpress ROS, and photodynamic therapy could be an effective therapeutic method. In this study, we established radioresistant cancer cells and examined the therapeutic effects and mechanisms with photodynamic therapy. We confirmed that X-ray-resistant cells showed overgeneration of mitochondrial ROS and elevated expression of HCP1, which led to the active accumulation of porphyrin and an increase in cytotoxicity with laser irradiation. Thus, photodynamic therapy is a promising treatment for X-ray-resistant cancers.

Keywords: HCP1; radioresistance; reactive oxygen species.

Figure 1

Cell viability after a single dose irradiation of X-ray was estimated and compared between the continual X-ray-irradiated RGK1 cells and the non-treated control cells by colony formation assay. Statistical significance was tested by Student’s t-test. n = 3, mean ± S.D., ** p < 0.01.

Figure 2

Intracellular ROS production level was estimated and compared between the X-ray resistant cells and the control cells. (A) representative images of cells stained with HPF and MitoTracker^TM Red CMXRos fluorescence dye. Scale bar: 50 μm. (B) the relative fluorescence intensity of HPF. (C) the relative fluorescence intensity of MitoSOX^TM Mitochondrial Superoxide Indicators. Statistical significance was tested by Student’s t-test. n = 120 for control samples in HPF experiment, n = 147 for X-ray resistant samples in HPF experiment, and n = 12 for MitoSOX^TM experiment. Mean ± S.D., ** p < 0.01.

Figure 3

The expression levels of HIF-1α and HCP1 in the X-ray-resistant cells and control cells were analyzed by Western blotting. (A) representative images of blotting bands. (B) the relative expressions of HCP1. (C) the relative expressions of HIF-1α. Statistical significance was tested by Student’s t-test. n = 10, mean ± S.D., * p < 0.05. The uncropped blots are shown in Figure S1.

Figure 4

Comparison of intracellular porphyrin accumulation levels by measurements of porphyrin fluorescence in cell lysates. Statistical significance was tested by Tukey’s HSD test. n = 6, mean ± S.D., ** p < 0.01.

Figure 5

Relative cell viability with or without laser irradiation at 2 J/cm² in the X-ray-resistant cells and the control cells. The viability is normalized with non-laser irradiated samples. Statistical significance was tested by Tukey’s HSD test. n = 6, mean ± S.D., ** p < 0.01.