Hematoporphyrin monomethyl ether-mediated sonodynamic therapy induces A-253 cell apoptosis

Abstract

It has been found that >90% of oral cancer patients suffer from squamous cell carcinoma (SCC). The 5-year survival rate of SCC is ~50%, despite the availability of different treatments. Sonodynamic therapy (SDT) has been developed as a novel therapy for cancer, resisting bacterial infection and inhibiting atherosclerotic plaque progression. The present study investigated the efficacy of hematoporphyrin monomethyl ether (HMME)-mediated SDT on the A-253 epidermoid cancer cell line. The cytotoxicity of HMME and the survival rate of cells following SDT were examined by the MTT assay. Apoptosis and necrosis of cells were detected using flow cytometry with Annexin V and propidium iodide (PI) staining, and fluorescence microscopy with Hoechst 33258 and PI staining. Intracellular reactive oxygen species (ROS) and Ca²⁺ levels were measured using a fluorescence microscope based on 2',7'-dichlorofluorescein diacetate and fluo-3/acetoxymethylester, respectively. Results of the MTT assay demonstrated that a lower concentration (<10 µg/ml) of HMME had no significant effect on the A-253 cells, but SDT combined with ultrasonic treatment for 1 min and 10 µg/ml HMME decreased the cell survival rate by 27%. Flow cytometry analysis revealed that A-253 cells in the SDT group had a higher rate of late apoptosis compared with the control group. Furthermore, fluorescence quantitation of apoptotic A-253 cells demonstrated that the percentages of apoptotic cells were increased in the ultrasound and SDT group compared with those in the control group. In the present study, the ROS level in the SDT group was elevated compared with that in the control group. The Ca²⁺ levels were increased to 181.2 and 268.7% in the ultrasound and SDT groups, respectively, relative to the control group. Taken together, the findings of the present study demonstrated that HMME-SDT significantly induces the apoptosis of A-253 cells together with intracellular ROS generation and Ca²⁺ overload. Thus, HMME-SDT may be a promising treatment option for patients with SCC.

Keywords: HMME; ROS; SCC; SDT; apoptosis.

Figure 1.

Schematic diagram of the ultrasonic generator and amplifier system used for sonodynamic therapy in vitro.

Figure 2.

Survival rate of A-253 cells following incubation with different HMME concentrations from 0–40 µg/ml, as determined using the MTT assay. Cells at the exponential growth phase were used in each experiment. Data are presented as the mean ± SD (n=6/group). *P<0.05 compared with the 0 µg/ml HMME group. HMME, hematoporphyrin monomethyl ether.

Figure 3.

A-253 cell survival rate following incubation with 10 µg/ml hematoporphyrin monomethyl ether plus ultrasound exposure time for 0, 1, 3, 5, 10 and 15 min. Data are presented as the mean ± SD (n=6/group). *P<0.05 compared with the 0 min ultrasound exposure time group.

Figure 4.

Apoptosis induced by HMME-SDT. (A) Apoptosis rates of A-253 cells measured using flow cytometry with Annexin V and PI double staining. The groups presented are the control, HMME (10 µg/ml), ultrasound (1 min) and SDT (10 µg/ml HMME, 1 min ultrasound exposure) groups. (B) Fluorescence photomicrographs of apoptotic and necrotic A-253 cells with Hoechst 33258 and PI double staining. Normal cells exhibited regular blue fluorescence. Apoptotic cells exhibited bright blue fluorescence (arrows) and necrotic cells exhibited pink fluorescence (arrowheads). Scar bar, 100 µm. (C) Fluorescence quantification of apoptotic and necrotic A-253 cells in control, ultrasound, HMME and SDT groups (n=6/group). *P<0.05 and **P<0.01 compared with the control group. HMME, hematoporphyrin monomethyl ether; SDT, sonodynamic therapy; PI, propidium iodide.

Figure 5.

Reactive oxygen species generation induced by SDT. (A) Fluorescence photomicrograph of A-253 cells stained by DCFH-DA. (B) Fluorescence intensity of intracellular ROS was measured by fluorospectrophotometry. Scar bar, 100 µm. Data are presented as the mean ± SD (n=6/group). **P<0.01 compared with the control group. HMME, hematoporphyrin monomethyl ether; SDT, sonodynamic therapy; DCFH-DA, 2′,7′-dichlorofluorescein diacetate.

Figure 6.

Ca²⁺ generation induced by SDT. (A) Fluorescence photomicrograph of A-253 cells stained by fluo-3/acetoxymethylester. (B) Fluorescence intensity of intracellular Ca²⁺ was measured by fluorospectrophotometry. Scar bar, 100 µm. Data are presented as the mean ± SD (n=6/group). *P<0.05 compared with the control group; **P<0.01 compared with the control group; ^#P<0.05 compared with the ultrasound group. HMME, hematoporphyrin monomethyl ether; SDT, sonodynamic therapy; Fluo-3 am, fluo-3/acetoxymethylester.