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This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
This study aimed to compare the effects of laser photodynamic therapy (PDT) with methylene blue (MB) or aminolevulinic acid (ALA) on the oral squamous cell carcinoma (OSCC) cell line.
In this in vitro experimental study, the C152 (KB) OSCC cell line was cultured in a culture medium containing 10% fetal bovine serum. The cells were exposed to 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM concentrations of MB and ALA alone and combined with diode laser irradiation with 660 nm wavelength, 40 mW power, and 10 J/cm 2energy density in continuous-wave mode perpendicular to the surface. Cell viability was assessed using the methyl thiazolyl tetrazolium assay and compared among the groups by the Kruskal–Wallis test.
The results showed that the reduction in cell viability in the MB + laser and ALA + laser groups was greater than that in the MB and ALA groups without laser (P < 0.001). Significant differences were noted in cell viability in the presence of some different concentrations of MB and ALA (P < 0.05), such that by an increase in their concentration, cell viability decreased. Cell viability in the MB + laser group was significantly lower than that in the ALA + laser group in some photosensitizer concentrations (P < 0.05).
Within the limitations of this in vitro study, the results showed that laser PDT with MB (high concentrations) was more effective than laser PDT with ALA against the OSCC cell line.
Cancer is a common cause of morbidity and mortality worldwide. Despite the significant advances made in cancer prevention, diagnosis, and treatment, a definite cure for cancer has not been identified yet, and it is still a common health dilemma worldwide. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the maxillofacial region. It can cause severe disfiguration and adversely affect patients' quality of life. It has been documented that tobacco use, high alcohol consumption, viral infections, malnutrition, chronic stimulation, poor oral hygiene, low intake of fruits and vegetables, and genetics are risk factors for it. Several treatment options such as surgical management, radiotherapy, and chemotherapy have been proposed for it with different successes and complications.
Photodynamic therapy (PDT) is a noninvasive treatment modality that involves the use of a dye as a photosensitizer (PS) and a suitable wavelength of light source that activates the PS. The PS binds to target cells, and it is activated by photons of the light source.
PDT was used in some studies for oral and head-and-neck cancers and premalignant lesions.
As a gap of information regarding the efficacy of PDT with aminolevulinic acid (ALA) against the OSCC cell line, this study aimed to compare the effects of laser PDT with methylene blue (MB) and ALA on the OSCC cell line. According to data searches, there was no similar study up to now.
This in vitro experimental study was conducted on KB cancer cell line (C152) purchased from the Pasteur Institute of Iran, Tehran, in 25 cm 2flasks (SPL, Germany). The study protocol was approved by the Ethics Committee of Islamic Azad University, Isfahan branch (IR.IAU.KHUISF.REC.1400.100). After the disinfection of flasks with 70% alcohol and observing cell density under an inverted microscope (Leica, Germany), they were incubated at 37°C with 5% CO 2and 95% humidity. The flasks were monitored daily in terms of cell proliferation and morphology and the absence of bacterial and fungal contamination. The culture medium of the flasks was refreshed every 1–2 days. For this purpose, under a laminar good (Airflow, Jal Tajhiz, Iran), the old culture medium and cells were transferred into sterile tubes and centrifuged at 1500 rpm for 5 min. A fresh culture medium containing 10% fetal bovine serum (Gibco, USA), bicarbonate buffer, amino acids, vitamins, 100 U/mL of penicillin, and 100 U/mL of streptomycin was then added to the tubes. When the cell density in the flasks reached over 85%, the cells were transferred into sterile Falcon tubes (SPL, Germany) and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, the fresh culture medium was added to the remaining cell sediment, and the suspension was administered among new flasks.
Cell counting
For cell counting, 50 μL of the cell suspension was mixed with 50 μL of 0.1% trypan blue (Merck, Germany) in phosphate-buffered saline, and the cells were counted using a Neubauer chamber. For this purpose, a certain volume of cell suspension was collected and dispensed under the Neubauer slide. Dead cells are stained with trypan blue, whereas the viable cells do not uptake it. Viable cells present in the four outer squares of the chamber were counted, and the mean number of viable cells was calculated. The number of cells in each milliliter of cell suspension was calculated using the following formula:
Number of cells/mL = Mean number of counted cells × 10 4× dilution coefficient.
Assessment of cytotoxicity by the methyl thiazolyl tetrazolium assay
For cytotoxicity assessment, 5 × 10 3cells were seeded in each well of 96-well plates (SPL, Italy) and incubated for 24 h. The cells were divided into five groups:
Group 1 – different concentrations of MB alone, Group 2 – different concentrations of ALA alone, Group 3 – different concentrations of MB with laser, Group 4 – different concentrations of ALA with laser, and Group 5 – controls without PS alone with/or laser. Eight wells were assigned to different concentrations of each PS.
The cells in Group 1 were subjected to 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM concentrations of MB (Merck, Germany), whereas the cells in Group 2 were exposed to 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM concentrations of ALA (Merck, Germany). As mentioned above, the cells in Groups 3 and 4 were subjected to different concentrations of MB and ALA, respectively, and received laser irradiation.
Laser irradiation
The diode LTR laser (Behsaz Gostar, Iran), 660 nm wavelength, 40 mW, 0 Hz, 250 s, 1 cm
2irradiation area, 10 J/cm
2, perpendicular and near contact to the plate and at room temperature was irradiated
Laser irradiation of cell culture medium.
Group 5 served as the control group. The control cells were not exposed to any PS and did not undergo laser irradiation.
The cells were then incubated for 24 h. After 24 h, the overlaying medium was removed, and the cells were rinsed with fetal bovine serum. Next, 5 μL of the methyl thiazolyl tetrazolium dye (5 mg/mL) was added to each well, and the plate was incubated at 37°C for 4 h. After 4 h, the culture medium was gently extracted from the wells by a syringe. For complete dissolution of formazan crystals in each well, 200 μL of dimethyl sulfoxide was added to each well, and then, the optical density of each well was read at 590 nm wavelength by a multimode microplate reader (BioTek, Synergy, USA), compared with the control group. The results were reported as the percentage of treated cells compared with the control cells using the following equation:
Percentage of viable cells = Mean optical density of treated cells/mean optical density of control cells × 100.
Data were analyzed using the Kruskal–Wallis test in SPSS version 26 software (IBM, Chicago, USA) and considering α = 0.05.
In each group, the Kruskal–Wallis test showed a significant difference in cell viability in the presence of different concentrations of MB or ALA (P < 0.001). Pairwise comparisons revealed that increasing the concentration of MB or ALA markedly decreased the viability of cells (P < 0.05). However, the difference was no longer significant when exposed to 1 mM to 10 mM concentrations of MB or ALA (P > 0.05).
Comparison of the four groups regarding the mean percentage of cell viability in the presence of different concentrations of methylene blue and aminolevulinic acid. ALA: Aminolevulinic acid.
By pairwise comparing of different concentrations of MB and ALA, it was showed that MB + laser had meaningful efficacy in reducing cell viability compared to MB alone (P < 0.001). ALA + laser had meaningful efficacy in reducing cell viability compared to ALA alone in 0.5 mM (P = 0.029) and 1 mM (P = 0.045). MB had meaningful efficacy in reducing cell viability compared to ALA in 0.2 mM (P = 0.025) and 2 mM (P = 0.012). MB + laser had meaningful efficacy in reducing cell viability compared to ALA + laser in 0.2 mM (P = 0.023) and 2 mM (P = 0.012).
PDT is a noninvasive modality for cancer treatment, which may be used alone or as an adjunct to surgery, chemotherapy, or radiotherapy. Following the activation of the PS, it undergoes a transition from a low-energy ground state to a higher energy state. This transition results in the formation of singlet oxygen, reactive oxygen species, and other reactive free radicals that are toxic to certain cells and microorganisms. Based on this selective cytotoxicity, PDT has been utilized in medicine for the treatment of a variety of conditions such as various cancers or microorganisms (photoactivated disinfection).
In the present study, MB was used as a PS due to its application for oral SCC.
The present results showed a significant difference in cell viability when treated with different concentrations of MB with or without laser. Minimum cell viability was recorded in a 10 mM concentration of MB. Furthermore, the reduction in cell viability was more significant in the MB + laser group than in MB alone. Like the present findings, Vahidi Banehkohal et al.
The effect of ALA in this study was similar to MB as minimum viability was recorded when oral carcinoma cells were treated with 10 mM ALA. Moreover, ALA + laser was more effective than ALA alone in reducing cell viability. Jerjes et al.,
Comparison of the mean cell viability in the four groups in escalating concentrations of PS s revealed significant differences. In some concentrations of PS, PDT with MB was more effective than ALA. As in these concentrations, MB alone was more effective than ALA, this efficacy may be related to PS than PDT.
According to this study, PDT with 10 mM MB and 10 J/cm 2660 nm diode laser had the best efficacy for killing vital OSCC cells, but as in different studies, different protocols were used, more studies to investigate the best treatment protocol for OSCC are necessary. In vivo studies also need for considering better treatment protocols for these patients.
In a 2022 study from Spain, a new PDT device was designed based on LED technology and used for four cases of premalignant and one superficial basal cell carcinoma with good results.
In a mini systematic review in 2022 for the evaluation of light-activated phytochemicals in PDT for cancer, some questions, such as most effective PS, its administration, the time of irradiation, light source, and sensitivity of cells toward PS, were considered for more evaluation.
PDT effects can be direct with destroying the tumoral mass or indirect effects such as vascular effects, apoptosis induction, inflammation, and generation of an immune response. This study only evaluated the direct effect of PDT, thus this is a limitation of this study, and in vivo, studies are needed to complete the evaluation of this technique.
To the best of our knowledge, this study was the first to compare the efficacy of PDT (660 nm diode laser) with MB and ALA against OSCC, which was a strength of this study. However, no previous study is available on this topic to compare our results with. Not assessing cell viability based on the duration of exposure of cells was another limitation of this study, which should be addressed in future studies. Maybe some studies are also required to assess some aspects of this treatment in animal models. Furthermore, the efficacy of different laser wavelengths and parameters and the long-term effects of this modality should be investigated.
Within the limitations of this in vitro study, the results showed that MB is better than ALA for reducing OSCC cell viability. PDT was better than each PS alone against OSCC cells. The MB + laser group was more effective than the ALA + laser group against the OSCC cell line in some concentrations. It may be related to the better efficacy of MB than ALA.
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Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.