Effects of meta-tetrahydroxyphenylchlorin photodynamic therapy on isogenic colorectal cancer SW480 and SW620 cells with different metastatic potentials

Abstract

The aim of this study is to investigate the antitumor effects and possible mechanisms of meta-tetrahydroxyphenylchlorin-mediated photodynamic therapy (m-THPC-PDT) on human primary (SW480) and metastatic (SW620) colon cancer cell lines. SW480 and SW620 cells were incubated with various concentrations of m-THPC, followed by photodynamic irradiation. Subcellular localization of m-THPC in cells was observed with confocal laser scanning microscopy (CLSM). Photocytotoxicity of m-THPC in the two cells was investigated by using MTT assay. The flow cytometry was employed to detect the cell apoptosis. The migration and long-term recovery ability were determined by scratch test and colony formation assay respectively. CLSM showed that m-THPC was mainly distributed within the endoplasmic reticulum (ER) and lysosome of SW480 cells and within the lysosome and mitochondria of SW620 cells. m-THPC-PDT induced a dose-dependent and light energy-dependent cytotoxicity in SW480 and SW620 cells. Apoptosis rate was approximately 65 and 25% in SW480 and SW620 respectively when the concentration of m-THPC increased to 11.76 μM. However, the rate of necrotic cells had no significant changes in two cell lines. The colony formation and migration ability of the two cell lines were decreased with m-THPC-PDT treatment in a dose-dependent manner. PDT with m-THPC not only could effectively inhibit cell proliferation and decrease migration ability and colony formation ability, but also could effectively kill SW480 and SW620 cells in a dose-dependent manner in vitro. These results suggest that m-THPC is a promising sensitizer that warrants further development and extensive studies towards clinical use of colorectal cancer.

Keywords: Apoptosis; Cell death; Colorectal cancer; Meta-tetrahydroxyphenylchlorin; Photodynamic therapy.

Fig. 1

Molecular structural formula of m-THPC

Fig. 2

Subcellular localization of m-THPC in SW480 (A–L) and SW620 (a–l) cells. Cells were incubated with 0.74 μM m-THPC for 8 h, then stained with 1 μmol/l endoplasmic reticulum marker (A–D: SW480, a–d: SW620), 50 nmol/l lysosome marker (E–H: SW480, e–h: SW620), and 100 nmol/l mitochondrion marker (I–L: SW480, i–l: SW620) at 37 °C for 30 min. Photographs were taken by confocal laser scanning microscopy. A, E, I, a, e, and i: m-THPC autofluorescence (red); B and b: endoplasmic reticulum marker (green); F and f: lysosome marker (blue); J and j: mitochondrion marker (green); C, G, K, c, g, and k: merged of A and B, E and F, I and J, a and b, e and f, and i and j, respectively; D, H, L, d, h, and l: phase contrast

Fig. 3

Cell viability of SW480 and SW620 determined by MTT assay after m-THPC-PDT. Two cell lines were incubated with 0–11.76 μM m-THPC for 8 h and then irradiated with 0–6.0 J/cm² light dose. The percentage of cell viability was shown in dose-response curve even 24 h after laser irradiation (a SW480, b SW620). Comparisons of cell viability in two cell lines with m-THPC at the same concentration and different light doses were demonstrated in c, d, and e (c 1.5 J/cm², d 3.0 J/cm², and e 6.0 J/cm²). All results are expressed as the mean ± SD of triplicate determinations from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the two cell lines

Fig. 4

Apoptosis assay using flow cytometry of SW480 and SW620 after m-THPC-PDT treatment. Cells were treated with 0–11.76 μM m-THPC, and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was performed after m-THPC-PDT. Representative flow cytometry figures are shown in A–C (SW480) and a–c (SW620)

Fig. 5

Cell migration ability of SW480 and SW620 after m-THPC-PDT treatment using scratch wound assay. The effects of different concentrations of m-THPC-PDT on cell migration are shown in A–E (SW480) and a–e (SW620). Phase contrast images were taken using the Olympus microscope at 48 h time point after m-THPC-PDT