Chlorophyllin e6‑mediated photodynamic therapy inhibits proliferation and induces apoptosis in human bladder cancer cells

Abstract

Patients with non‑muscle invasive bladder cancer (NMIBC) frequently relapse following surgery due to incomplete resection and chemoresistance, highlighting the importance of developing novel therapeutic strategies that mechanistically assist in eradicating the residual tumor. The aim of the present study was to evaluate the anticancer effect of chlorophyllin e6‑mediated photodynamic therapy (e6‑PDT) and its potential mechanisms by using monolayer cells or multicellular tumor spheroid models of human bladder cancer cells (T24 and 5637). The results revealed that e6‑PDT exhibited significant cytotoxicity in the T24 and 5637 cells of these two models as detected by the Water‑Soluble Tetrazolium Salts‑1 and CellTiter‑Glo Luminescent Cell Viability assays, respectively. Cell migration and invasion capacities decreased markedly following e6‑PDT. In addition, the cells following e6‑PDT exhibited typical morphological changes of apoptosis as detected by fluorescence microscopy with 4',6‑diamidino‑2‑phenylindole staining and transmission electron microscopy. A greater number of apoptotic cells were observed post‑e6‑PDT by flow cytometry. The expression levels of poly(adenosine diphosphate‑ribose) polymerase (PARP) and B‑cell lymphoma 2 protein were decreased, while cleaved PARP was increased, significantly following e6‑PDT as determined by western blotting. The level of intracellular reactive oxygen species (ROS) was increased, while the activity of superoxide dismutase (SOD) was decreased, significantly in e6‑PDT‑treated cells. Thus, the novel e6‑PDT exhibits prominent photo‑cytotoxicity effect and the induction of apoptosis was probably due to the inhibition of SOD activity and the generation of ROS. These results indicate that chlorophyllin e6 is an effective photosensitizer and that e6‑PDT may have a therapeutic application for the treatment of bladder cancer.

Figure 1.

Phototoxic effect of e6-PDT in monolayer cells of T24 and 5637. (A and B) The morphological changes of T24 and 5637 cells were observed using an inverted microscope at 24 h following e6-PDT. Monolayer cells in the e6-PDT group exhibited cell shrinkage, vacuole formation and cell detachment from each other. Scale bar=25 µm. (C and D) Cell viability of T24 and 5637 monolayer cells was measured using the Water Soluble Tetrazolium salts-1 assay (n=3). **P<0.01 and ***P<0.001 vs. blank control group. e6-PDT, chlorophyllin e6-mediated photodynamic therapy.

Figure 2.

Phototoxic effect of e6-PDT in T24 and 5637 MCTSs. (A and B) Morphological changes of T24 and 5637 MCTSs were recorded using an inverted microscope at 24 h post-e6-PDT. MCTSs in the e6-PDT group exhibited destruction of spheroid structure and density reduction. Scale bar=100 µm. (C and D) Cell viability of T24 and 5637 MCTSs was determined by the CellTiter-Glo assay (n=3). **P<0.01 and ***P<0.001 vs. blank control group. e6-PDT, chlorophyllin e6-mediated photodynamic therapy; MCTSs, multicellular tumor spheroids.

Figure 3.

Inhibitory effect of e6-PDT on bladder cancer cell proliferation. (A) A cell colony formation assay was performed. (B and C) Effect of e6-PDT treatment on the long-term proliferation of T24 and 5637 cells was evaluated (n=4). *P<0.05, **P<0.01 and ***P<0.001 vs. blank control group. e6-PDT, chlorophyllin e6-mediated photodynamic therapy.

Figure 4.

Inhibitory effect of e6-PDT on the migration and invasion of bladder cancer cells. A wound healing assay was applied to measure the migration ability of (A and B) T24 and (C and D) 5637 cells following e6-PDT. Images were captured at the 0 and 24 h time points and the widths of each wound were measured. Scale bar=500 µm. (E) Cell invasion capacity of (F) T24 and (G) 5637 cells was evaluated using a Transwell assay. Scale bar=100 µm. Migrated cells were photographed and quantified following 48 h (n=3). **P<0.01 and ***P<0.001 vs. blank control group. e6-PDT, chlorophyllin e6-mediated photodynamic therapy.

Figure 4.

Inhibitory effect of e6-PDT on the migration and invasion of bladder cancer cells. A wound healing assay was applied to measure the migration ability of (A and B) T24 and (C and D) 5637 cells following e6-PDT. Images were captured at the 0 and 24 h time points and the widths of each wound were measured. Scale bar=500 µm. (E) Cell invasion capacity of (F) T24 and (G) 5637 cells was evaluated using a Transwell assay. Scale bar=100 µm. Migrated cells were photographed and quantified following 48 h (n=3). **P<0.01 and ***P<0.001 vs. blank control group. e6-PDT, chlorophyllin e6-mediated photodynamic therapy.

Figure 5.

Morphological and ultrastructural changes of apoptosis. (A) Morphological alterations of the apoptotic nuclei of T24 monolayer cells following e6-PDT were assessed using DAPI staining. Scale bar=50 µm. (B) Ultrastructural changes of T24 cells, monolayer cells and MCTSs, following e6-PDT were observed using TEM. (C) Morphological alterations of the apoptotic nuclei of 5637 monolayer cells following e6-PDT were assessed using DAPI staining. Scale bar=50 µm. (D) Ultrastructural changes of 5637 cells, monolayer cells and MCTSs, following e6-PDT were observed and compared by TEM. DAPI staining revealed karyopyknosis and nuclear fragmentation in the e6-PDT group. TEM revealed chromatin condensation and edge accumulation (thick arrows), mitochondria swelling (thin arrows), and cytoplasm vacuolization (arrowheads) in the e6-PDT group (scale bar=5 µm on a left image and 500 nm on a right image in each cell type). e6-PDT, chlorophyllin e6-mediated photodynamic therapy; TEM, transmission electron microscopy; DAPI, 4′6-diamidino-2-phenylindole.

Figure 6.

Induction of apoptosis by e6-PDT in bladder cancer cells. (A) Apoptotic rate of (B) T24 and (C) 5637 cells following e6-PDT was quantified by flow cytometry with Annexin V-FITC/PI double staining (n=3). **P<0.01 and ***P<0.001 vs. blank control group. Western blot analysis was performed to detect the expression of the apoptosis-associated proteins PARP, cleaved PARP and Bcl-2 in (D) T24 and (E) 5637 cells at 2, 12 and 24 h post-treatment. e6-PDT, chlorophyllin e6-mediated photodynamic therapy; PARP, poly(adenosine diphosphate-ribose) polymerase; Bcl-2, B-cell lymphoma-2; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 7.

Detection of ROS and SOD in bladder cancer cells following e6-PDT. (A) DCFH-DA reagent was used to measure the production of intracellular ROS following e6-PDT. Cells with DCF fluorescence were examined with flow cytometry. (B and C) Quantification of ROS generated in T24 and 5637 cells (n=3). (D and E) SOD activity of T24 and 5637 cells following e6-PDT was determined using a SOD Assay Kit-WST (n=3). The inhibition rate of WST reflects the activity of SOD. **P<0.01 and ***P<0.001 vs. blank control group. ROS, reactive oxygen species; SOD, superoxide dismutase; e6-PDT, chlorophyllin e6-mediated photodynamic therapy; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; WST, Water-Soluble Tetrazolium.

Figure 8.

A schematic diagram of e6-PDT induces apoptosis in human bladder cancer cells. Following e6-PDT treatment, the activity of intracellular antioxidant enzyme SOD is reduced, which lead to the improvement of ROS generation in the mitochondria. An increased ROS activates pro-apoptotic proteins and inhibits an anti-apoptotic protein Bcl-2, leading to the release of Cyto-c from the mitochondria to the cytoplasm. Cyto-c along with Apaf-1 can cause the activation of caspase-9, which in turn activates the downstream effector caspases such as caspase-3 and caspase-7. The activated caspase-3 and caspase-7 further initiate the cleavage of downstream nuclear protein PARP, thus inducing cell apoptosis. e6-PDT, chlorophyllin e6-mediated photodynamic therapy; SOD, superoxide dismutase; ROS, reactive oxygen species; Bcl-2, B-cell lymphoma-2; Cyto-c, cytochrome c; Apaf-1, apoptosis activating factor-1; PARP, poly(adenosine diphosphate-ribose) polymerase.