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Introduction

Globally in 2018, >90% of oral cancer patients suffer from squamous cell carcinoma (SCC), a malignancy potentially associated with lymph node metastasis (1). SCC starts with variation in mucosal epithelial cells and results in cells with the essential capabilities for malignant growth, loss of cell cycle control and spread (2). Furthermore, the epithelial dysplasia that occurs in SCC reduces the adhesion between cells, which facilitates separation of cells from the tumor and allows metastasis to other organs (3). The 5-year survival rate of SCC is ~50% despite the availability of numerous treatment options, including radiotherapy, chemotherapy and surgical excision (4). A new promising minimally invasive treatment method for patients with SCC is needed due to the damage to the human body, the unreliable efficacy and the side effects of traditional clinical treatments (5).

Sonodynamic therapy (SDT) is an alternative approach for treating patients with SCC that utilizes the synergistic effects of low-intensity ultrasound and sonosensitizers to kill cancer cells, resist bacteria and inhibit atherosclerotic plaque progression (6–8). Hematoporphyrin monomethyl ether (HMME) is an effective sonosensitizer in SDT with a stable structure, lower dark toxicity and higher singlet oxygen yield to induce cell apoptosis via the mitochondrial apoptotic pathway (9). Calcium ion (Ca2+) is a secondary messenger which can regulate apoptosis and a sudden increase of intracellular Ca2+ can induce oxidative stress inside the cells (10). During the process of SDT, the sonosensitizer is activated releasing reactive oxygen species (ROS) that are the principle mediators of cell apoptosis (11,12). However, uncontrolled ROS activity in cells may induce the release of ROS by adjacent mitochondria initiating a positive feedback loop resulting in excessive ROS production leading to mitochondrial injury (13).

Studies have also demonstrated that excessive ROS levels generated during SDT stimulate apoptotic signaling pathways, which include the proteins caspase-3, caspase-9 and Bax (14). During SDT, cell membrane integrity is destroyed together with the loss of the mitochondrial membrane potential (MMP) (15,16). This MMP loss leads to mitochondrial membrane permeabilization (14) and the release of cytochrome c, which activates caspase-9, followed by caspase-3 and caspase-7 (17). Some studies have demonstrated that following protoporphyrin IX mediated SDT, human tongue squamous carcinoma SAS cells are arrested at the G2/M phase of the cell cycle and upregulate p53, which can activate the Fas apoptosis pathway eventually leading to cell death (18,19).

The present study investigated the effect of HMME-mediated SDT on A-253 cells The findings of the present study may facilitate the quest for a promising alternative approach for treating patients with SCC.

Materials and methods

Cell culture

The A-253 cell line is derived from a human submandibular gland epidermoid carcinoma (20). Human SCC A-253 cells were purchased from the American Type Culture Collection and cultured in modified McCoy's 5a medium (American Type Culture Collection) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA.) in an incubator containing 5% CO2 at 37°C.

Treatment of cells with HMME

The A-253 cells were incubated in 96-well plates with different concentrations of HMME (0–40 µg/ml at 5 µg/ml intervals) for 90 min in the dark at 37°C. The sterile HMME solution was supplied by Shanghai Xianhui Pharmaceutical Co., Ltd.

MTT assay

To investigate the cytotoxicity of HMME, cell viability was measured using the MTT assay. Cells at the exponential growth phase were used in each experiment. Overall, 10 µl MTT (5 mg/ml) was added to each well and incubated at 37°C incubator for 4 h. After removal of the MTT, 1 ml DMSO was added to dissolve the violet formazan crystals. Absorbance at 570 nm was measured using the microplate reader Bio-Tek ELX800 (Biotek Instruments, Inc.).

SDT treatment in vitro

Cell suspension was added to a 3.5-mm petri dish and later positioned in the tank of degassed distilled water ensuring that the cells were 3 cm away from the ultrasound transducer (Fig. 1). The ultrasonic generator, amplifier and transducer utilized in this experiment were supplied by the Harbin Institute of Technology. The ultrasound resonance frequency was 1 MHz, with 30% duty factor and 100 Hz repetition rate, and the ultrasound intensity was 1.5 W/cm2 as measured by a needle hydrophone (HNC-1000; Onda Corp) inside the well. The temperature change in the medium should not be >1°C during the experiment. In the present study, MTT results demonstrated that 10 µg/ml HMME was the optimal concentration to use for in vitro SDT treatment. HMME (10 µg/ml) was then applied in combination with different ultrasonic durations (0, 1, 3, 5, 10 and 15 min) to investigate the survival rate of A-253 cells.

Figure 1. Schematic diagram of the ultrasonic generator and amplifier system used for sonodynamic therapy in vitro.

Cell apoptosis and necrosis analysis

To investigate cell apoptosis and necrosis following SDT, the Annexin V-FITC apoptosis kit (Merck KGaA) was used for flow cytometry analysis according to the manufacturer's instructions. Subsequently, cells in McCoy's 5a medium were randomly divided into 4 groups: The control group (PBS), the ultrasound group (1 MHz; 1.5 W/cm2), the HMME group (10 µg/ml) and the SDT group (1 MHz; 1.5 W/cm2 ultrasound combined with 10 µg/ml HMME). After incubation with HMME or PBS for 90 min at 37°C, both the ultrasound group and the SDT group were exposed to ultrasound for 1 min at room temperature. After 3 h of treatment, 1×106/ml cell per well were then incubated with 5 µl Annexin V and 5 µl PI (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature in the dark. Samples were detected using a FACSCalibur flow cytometer (BD Biosciences). FlowJo version 10 (Tree Star, Inc.) was used to analyze data.

Cell apoptosis and necrosis were also investigated using Hoechst 33258 and PI staining (Merck KGaA) followed by fluorescence microscopy. Firstly, the treated samples were washed with PBS 3 times and then stained according to the manufacturer's protocols. In brief, samples were incubated with 10 µg/ml PI for 10 min at 37°C in the dark and stained with 5 µg/ml Hoechst 33258 (Merck KGaA)for 5 min. Following incubation, the samples were washed with PBS twice and analyzed using the fluorescence microscope (Zeiss GmbH) at the excitation wavelength of 330–385 nm and the emission wavelength of 420–480 nm at ×200 magnification.

Intracellular ROS and Ca2+ measurements

The A-253 cells were incubated with 20 µΜ 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Merck KGaA) and 10 µM fluo-3/acetoxymethylester (Μerck KGaA) for 30 min at 37°C to measure the fluorescence of intracellular ROS and Ca2+, respectively. Samples were then washed 3 times with PBS and immediately surveyed using the fluorescence microscope. A total of 1×106 cells were collected and measured using a fluorospectrophotometer (Varian Australia Pty, Ltd.) at 488 nm excitation and 525 nm emission wavelengths.

Statistical analysis

All experiments were repeated three times independently and all values were expressed as the mean ± standard deviation. Differences between multiple groups were analyzed using one-way ANOVA, followed by Tukey's post-hoc test. Statistical analyses were performed using SPSS version 25.0 (IBM, Corp). P<0.05 was considered to indicate a statistically significant difference.

Results

Increasing HMME concentration decreases A-253 cell survival rates

Cytotoxicity of HMME was measured using the MTT assay. Fig. 2 represents the A-253 cell survival rates at different HMME concentrations. With increasing HMME concentration, the cell survival rate decreased. The lowest cell survival rate was 71% when 40 µg/ml HMME was used. However, HMME concentrations of 5 and 10 µg/ml did not have significant effects on cell survival rates.

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.

Increased ultrasound exposure time during SDT decreases A-253 cell survival rates

Cell survival rates after SDT treatment was also detected by MTT assay. As represented by Fig. 3, the survival rates of A-253 cells following SDT combined with HMME treatment depended on the duration of ultrasound exposure. When the ultrasound exposure time was increased from 1 to 15 min, the survival rate was significantly decreased from 73 to 62% (Fig. 3).

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.

Apoptosis is induced by SDT

Flow cytometry results are represented in Fig. 4A. Cells in the right lower quadrant (Annexin V+/PI−) represent early apoptotic cells, those in the right upper quadrant(Annexin V+/PI+) represent late apoptotic cells. The apoptosis rates of A-253 cells were as follows: SDT treatment (32.10%), HMME (8.01%) and ultrasound (22.50%) compared with the control group (5.73%) (Fig. 4A). In the double staining with Hoechst 33258 and PI, normal cells displayed regular blue fluorescence, apoptotic cells bright blue fluorescence and necrotic cells pink fluorescence (Fig. 4B). The percentages of apoptotic cells were increased to 14.6% in the ultrasound group (P<0.05) and 22.8% in the SDT group (P<0.01), compared with those in the control group (Fig. 4C). However, the apoptotic rate in the HMME group demonstrated no significant difference compared with the control group. According to the results of fluorescence quantification, very few necrotic cells were identified in all groups (Fig. 4C). Taken together, these results demonstrate that the rate of apoptosis in the A-253 cell line was increased by SDT.

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.

ROS generation increases with SDT

DCFH-DA fluorescence was observed mainly in the SDT group compared with the other 3 groups (Fig. 5A). The ROS level in the SDT group was significantly increased (363.1%; P<0.01), while showing no significant difference in the ultrasound (113.6%; P>0.05) and the HMME (92.5%; P>0.05) groups compared with that in the control group (Fig. 5B).

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.

Calcium overload in A-253 cells treated using SDT and ultrasound

Calcium fluorescence intensity was observed more in the SDT group and to a lower extent in the ultrasound group compared with that in the control group (Fig. 6A). Furthermore, Ca2+ levels were increased to 181.2% (P<0.05) and 268.7% (P<0.01) in the ultrasound and SDT groups, respectively, compared with those in the control group (Fig. 6B). Together these results demonstrate that Ca2+ levels were significantly increased in A-253 cells following SDT and ultrasound.

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.

Discussion

HMME, a second-generation sonosensitizer, has been extensively utilized in SDT treatment due to its low toxicity and high selectivity by highly metabolic tissues (21). The optimal concentration of HMME used in the present study is consistent with the results of a previous study that reported the highest cytotoxicity of SDT to U937 cells in the presence of 10 µg/ml HMME (6). Hao et al (10) also reported the lowest viability of C6 cells in the presence of 1 MHz ultrasound combined with 10 µg/ml HMME.

It is well documented that apoptosis is the major form of death in numerous types of cancer cells in response to SDT (22,23), which is in accordance with the results of the current study. In the present study, the apoptotic rate in the SDT group was 32.10% (P<0.05), while the rates in the HMME and the ultrasound treatment groups were 8.01 and 22.50%, respectively. In addition, the Hoechst 33258 and PI assays confirmed the results of the flow cytometry indicating that the numbers of apoptotic cells were increased in the SDT group compared with those in other groups. During the SDT process, the sonosensitizer is activated and ROS is released; the imbalance between ROS release and elimination may induce further ROS release by the mitochondria (13). This positive feedback results in excess ROS production resulting in mitochondrial injury and apoptosis (24). The ROS level was significantly increased in the SDT group but not in the ultrasound and HMME groups, compared with that in the control group. The findings of the present study indicated that HMME-SDT enhances ROS release and affects cellular conditions of A-253 cells. Notably apoptosis fluorescence was also observed in the ultrasound group confirming previous findings (25). It is well known that ultrasound alone can exert acoustic streaming and cavitation, thereby inducing various biological effects such as exerting shear stresses on the cell membrane, pore formation and endocytosis, leading to induction of cell apoptosis (26).

Ca2+ serves a key role as a second messenger in cellular transmission (27). Intracellular Ca2+ overload may induce cell apoptosis or death (10). As a result, high intracellular Ca2+ levels can be regarded as a signal of early apoptosis (28). The findings of the present study demonstrated that the Ca2+ levels were increased in the ultrasound and SDT groups compared with those in the control group. During the process of SDT, cavitation can also occur. When the cell membrane is broken, molecules such as Ca2+ can enter the cell by passive diffusion (29). ROS overload may regulate ion channels, including the Ca2+ channel, which also induces Ca2+ influx (30). These findings may explain the phenomenon of Ca2+ overload in both the SDT and ultrasound groups in the current study.

In conclusion, HMME-SDT significantly induces apoptosis, leading to ROS generation and Ca2+ overload in A-253 cells. HMME-SDT may be a promising alternative approach in patients with SCC.

Acknowledgements

Not applicable.

Funding

This study was supported by grants from the Natural Science Foundation, Heilongjiang Province of China (no. H2017022) and the Natural Science Foundation of China (no. 81670994).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YZ and DZ conceived, designed and performed the experiments. ZH, WC and LB analyzed the data. YZ wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References