Sinoporphyrin sodium is a promising sensitizer for photodynamic and sonodynamic therapy in glioma

Abstract

The aim of the present study was to explore the antitumor effects of sinoporphyrin sodium (DVDMS)‑mediated photodynamic therapy (PDT) and sonodynamic therapy (SDT) in glioma, and to reveal the underlying mechanisms. The uptake of DVDMS by U‑118 MG cells was detected by flow cytometry (FCM). A 630‑nm semiconductor laser and 1‑MHz ultrasound were used to perform PDT and SDT, respectively. Cell proliferation and apoptosis were evaluated using the Cell Counting Kit‑8 assay, FCM and Hoechst 33258 staining, respectively. Western blot analysis was used to detect protein expression and phosphorylation levels. BALB/c nude mice were used to establish a xenograft model of U‑118 MG cells. DVDMS was injected intravenously and PDT and SDT were performed 24 h later. An in vivo imaging system was used to evaluate the fluorescence of DVDMS, to measure tumor sizes, and to evaluate the therapeutic effects. The uptake of DVDMS by U‑118 MG cells was optimal after 4 h. PDT and SDT following DVDMS injection significantly inhibited the proliferation and increased apoptosis of glioma cells in vitro (P<0.05, P<0.01) respectively. In vivo, the fluorescence intensity of DVDMS was lower in the PDT and SDT groups compared with the DVDMS group, while tumor cell proliferation and weight were lower in the PDT and SDT groups than in the control group (P<0.05, P<0.01). However, there was no significant difference when laser, ultrasound or DVDMS were applied individually, compared with the control group. Hematoxylin and eosin staining suggested that both PDT and SDT induced significant apoptosis and vascular obstruction in cancer tissues. DVDMS‑mediated PDT and SDT inhibited the expression levels of proliferating cell nuclear antigen (PCNA) and Bcl‑xL, increased cleaved ‑caspase 3 levels, and decreased the protein phosphorylation of the PI3K/AKT/mTOR signaling pathway. Changes in the expression of PCNA, and Bcl‑xL and in the levels of cleaved‑caspase 3 were partly reversed by N‑acetyl‑L‑cysteine, a reactive oxygen species (ROS) scavenger. Similar results were obtained with FCM. DVDMS‑mediated PDT and SDT inhibited glioma cell proliferation and induced cell apoptosis in vitro and in vivo, potentially by increasing the generation of ROS and affecting protein expression and phosphorylation levels.

Keywords: sinoporphyrin sodium; photodynamic therapy; sonodynamic therapy; glioma; protein expression and phosphorylation.

Figure 1.

Uptake of DVDMS by U-118 MG cells. (A) The absorption spectrum of DVDMS showed five absorption peaks at 369, 517, 550, 577 and 631 nm, respectively. (B) Cellular uptake after different incubation times (0, 2, 4 and 6 h). The cell density of U-118MG was 10⁶ cells/ml and the concentration of DVDMS was 8 µM. Uptake was measured using the APC channel (with excitation and emission wavelengths of 633 and 660 nm, respectively). DVDMS, sinoporphyrin sodium.

Figure 2.

Effects of DVDMS-mediated PDT and SDT on proliferation and apoptosis of U-118 MG cells. (A) Inhibition of cell proliferation, *P<0.05, **P<0.01, ***P<0.001 vs. DVDMS. (B) Hoechst 33258 staining of apoptotic cells: (B₁) control group, (B₂) laser group, (B₃) ultrasound group, (B₄) DVDMS group, (B₅) PDT group, (B₆) SDT group; original magnification was ×100, and the yellow bar indicates 100 µm. (C) Effects of different treatment times on apoptosis, **P<0.01 vs. the control group. (D) Effects of NAC on PDT- and SDT-induced cell apoptosis: (D₁) control group, (D₂) DVDMS group, (D₃) PDT group, (D₄) PDT+NAC group, (D₅) SDT group, (D₆) SDT+NAC group. DVDMS, sinoporphyrin sodium; PDT, photodynamic therapy; SDT, sonodynamic therapy; NAC, N-acetyl-L-cysteine.

Figure 3.

In vivo antitumor effects of DVDMS-mediated PDT and SDT in a xenograft mouse model. (A) In vivo fluorescence measurement. The DVDMS dose in the three groups was 2.00 mg/kg. (B) Representative ultrasound images of a xenograft tumor. (C) Ideograph of DVDMS-mediated PDT and SDT. (D) Comparison of tumor size (cm²) and weight (g). The DVDMS dose in the low-dose groups and high-dose groups was 1.0 and 2.0 mg/kg, respectively. PDT was performed using a 630-nm laser with a luminous flux of 150 mW/cm² for 10 min. SDT groups received ultrasonic irradiation at 1.00 MHz with an intensity of 500 mW/cm² for 3 min. **P<0.01 vs. the control group. DVDMS, sinoporphyrin sodium; PDT, photodynamic therapy; SDT, sonodynamic therapy.

Figure 4.

DVDMS-mediated PDT and SDT inhibition of the PI3K/AKT/mTOR signaling pathway. (A) PCNA, (B) Bcl-xL, (C) Bax, (D) p-PI3K, (E) p-AKT, (F) p-mTOR, (G) p-p70s6K, (H) p-rps6, (I) p-4EBP-1, (J) p-eIF4E, (K) p62, (L) LC3-II/LC3-I. Concentrations of DVDMS in the three groups were 0, 0.03, 0.13 and 0.5 µM. PDT and SDT were performed 4 h after co-incubation, n=3, *P<0.05, **P<0.01 vs. DVDMS. DVDMS, sinoporphyrin sodium; PDT, photodynamic therapy; SDT, sonodynamic therapy; PCNA, proliferating cell nuclear antigen; Bcl-xL, B-cell lymphoma-extra large; Bax, BCL2-associated X; p-PI3K, phospho-phosphatidylinositol 3 kinase; p-AKT, phospho-protein kinase B; p-mTOR, phospho-mammalian target of rapamycin; p-p70s6k, phospho-p70 ribosomal s6 kinase; p-rps6, phospho-ribosomal protein s6; p-4EBP1, phospho-4E binding protein 1; p-eIF4E, phospho-protein synthesis initiation factor 4E; SQSTM1/p62, sequestosome 1; LC3, light chain 3.