Photodynamic Therapy Effects on Oral Dysplastic Keratinocyte Cell Cultures: A Systematic Review

Abstract

Photodynamic therapy (PDT) represents a therapeutic intervention applied in various pre-malignant and malignant disorders’ treatments. The interaction between a photosensitizer (PS), ideal wavelength radiation, and tissue molecular oxygen activates a series of photochemical reactions liable to produce reactive oxygen species. These highly reactive species allow for the decrease cell proliferation and yield cancerous and pre-cancerous cell death. The aim of this work is to carry out a systematic review to investigate the effects of in vitro PDT for oral potential malignant disorders (OPDM) cell lines. This systematic review was conducted according to the PRISMA protocol, and the PROSPERO registration number was CRD42022362349. An electronic search was performed on the following search engines: PubMed, Embase, and Web of Science. The Population, Intervention, Comparison, Outcomes, and Study design (PICOS) has been applied as the method by which to outline our study eligibility criteria. The QUIN tool was employed to interpret the risk of bias of the included studies. Initially, seventy-five records were retrieved through databases, and after the selection steps, seven items finally met our inclusion criteria. The preliminary search resulted in 75 studies, out of which 22 were found to be duplicates. After reviewing the titles and abstracts of the remaining 53 studies, 45 were rejected as they did not meet the inclusion criteria. Further evaluation of the full texts led to the exclusion of only one article, since the full text was not available. As a result, seven studies were ultimately identified and included in the analysis. The main findings confirm the role of in vitro photodynamic therapy using several photosensitizers as a potential treatment for oral potentially malignant disorders.

1. Introduction

Topical photodynamic therapy (PDT) is a non-invasive approach involving a photosensitizer, light source, and oxygen to selectively produce a cytotoxic effect on targeted tissue [1]. The several combinations of a light source and photosensitizer allow this technique to target different lesions [2]. The molecular mechanism of PDT is based on three elements: the photosensitizer (PS); light with the appropriate wavelength; and dissolved oxygen in the cells [3]. These three compounds create the desired results within pathological tissues through mutual interactions. Photosensitizer molecules absorb light of the proper wavelength, starting the activation processes that leads to the death of selected cells [4]. The main photosensitizers studied in oral medicine are 5-aminolevulinic acid (5-ALA), methylene blue, and toluidine blue, and the wavelength of the light source can vary from 600 to 800 nm [5,6].

PDT has been used in diverse specialties from ophthalmology to gynecology, including diagnostics and numerous dental domains such as endodontics, restorative dentistry, periodontology, and oral medicine [7,8,9,10,11,12]. In this field, PDT has been increasingly studied for its potential role in the treatment of oral potentially malignant disorders (OPMDs) such as oral leukoplakia (OL) and oral lichen planus (OLP) [13,14,15]. OPMDs are disorders with a high risk of progression to oral squamous cell carcinoma (OSCC). During the 2005 World Health Organisation (WHO) workshop, the terminology, definitions, and classification of oral lesions with a predisposition to malignant transformation were discussed. The term “potentially malignant disorders” was suggested. OPMDs include a group of conditions involving the oral mucosa with a raised risk of malignancy and comprised oral leukoplakia, erythroplakia, erythroleukoplakia, and proliferative verrucous leukoplakia (PVL). Although malignant degeneration is the most feared and meaningful outcome, fortunately, not all lesions progress to that stage. They may last unchanged, broaden or reduce in size, or resolve completely. Presently, the degree of oral epithelial dysplasia (OED) is directly related to malignant transformation. Most authors agree that the risk of progression rises with the degree of OED. Indeed, most literature corroborates that OED has a significant risk of malignant transformation and should be considered in the clinical risk management process of OPMD progression [3,16,17].

The in vitro models provide a controlled environment for evaluating the phototoxicity of PDT agents, light sources, and treatment conditions. PDT has shown a cytotoxic effect due to the production of reactive oxygen species (ROS), microvascular alteration, and the activation of the immune system [18]. In vitro PDT also allows for the study of the molecular changes that occur in response to PDT, providing valuable proof to assess the treatment’s biological effects [19]. The ability of PDT to selectively target lesional tissue allows the clinician to improve the prognosis of patients with OPMDs significantly [20]. Moreover, in vitro PDT can provide valuable information on the optimal treatment parameters, including photosensitizer concentration, light dose, and treatment time, which can be used to optimize in vivo PDT treatments [21]. However, there is still much to be learned about the mechanisms and limitations of in vitro PDT, and continued research in this area is crucial to realize the full potential of PDT for the treatment of OPMDs and other oral diseases. Cell culture models provide a controlled environment for evaluating the phototoxicity of PDT agents, light sources, and treatment conditions, and they allow for the exploration of the molecular changes that happen in response to PDT [22]. In vitro, PDT also provides valuable information on the optimal treatment parameters, including photosensitizer concentration, light dose, and treatment time, which can be used to optimize in vivo PDT treatments. The in vitro PDT studies of OPMDs have significantly contributed to the development and optimization of this promising treatment approach. The site of localization of a PS is usually the primary target for PDT since the ROS produced due to the PDT have a very short lifetime (0.05 ms) [23]. Since keratinocytes of the most superficial layer of oral mucosae are, therefore, the leading target site of cytotoxic phenomena caused by PDT, and oral epithelial dysplasia is still the most important prognostic factor for malignant transformation, the purpose of this review is to evaluate the effects of in vitro PDT on dysplastic keratinocyte cell cultures.

2. Materials and Methods

A systematic literature search was performed using a different database (PubMed, the Cochrane Library, and ISI Web of Science) following the PRISMA checklist. The search operations ended on 28 October 2022. The search terms used (MeSH terms) were “(“photodynamic therapy”) AND (“in vitro” OR “cell culture”) AND (“oscc” OR “oral squamous cell carcinoma” OR “opmd” OR “oral potentially malignant disorders” OR “oral pre-cancerous”)”. The flowchart in Figure 1 shows all the results. A structured approach was used to formulate the research question using five components (“PICOS”): the study population (P) consists of dysplastic cell culture treated by topical photodynamic therapy; the intervention (I) was in vitro photodynamic therapy; the comparison (C) was no treatment; the outcome (O) was to assess the results of topical photodynamic therapy in the treatment of pre-malignant and malignant lesions; and the study designs (S) included cross-sectional studies, retrospective cohort studies, prospective comparative studies, case-control studies, case series, and case report. The selection took different steps: after collecting all the initial results, three reviewers (D.D.S., A.L., and A.R.) read the titles and abstracts, excluded duplicates, and ruled out all those articles that did not meet the inclusion criteria during this initial analysis. Then, two reviewers (D.D.S. and A.L.) read the full texts of the remaining articles in depth to better evaluate the content.

The inclusion criteria were as follows:

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In vitro studies regarding PDT and potentially or frankly malignant conditions;

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Abstract and main text in English;

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Studies published since 1990.

The exclusion criteria were as follows:

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In vivo or ex vivo studies;

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Full text not available;

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Reviews, Acts of Congress, and Letters to the Editor.

The quality of the included studies was assessed with the QUIN tool (risk-of-bias tool for assessing in vitro studies conducted in oral medicine [23]. Twelve bias domains were assessed, and final assessment was performed by categorizing each of the studies as having “low”, “medium”, or “high” risk of bias. D.D.S. and A.R. performed the bias analysis independently; a third author (A.L.) refereed the conflicts. Data were extracted from the included studies using a standardized data extraction form. Extracted data included the following:

• Study characteristics: author, publication year, study design, and sample size;
• Photodynamic therapy parameters: light source, photosensitizer used, concentration, exposure time, and irradiance;
• Outcome measures: cell viability, apoptosis, necrosis, reactive oxygen species generation, and other relevant endpoints.

Any discrepancies in data extraction were resolved through discussion and consensus between the three reviewers. Data were synthesized due to the anticipated heterogeneity of the included studies. Results from individual studies were summarized and presented descriptively. Key findings, trends, and limitations of the included studies were discussed. As this systematic review focused on in vitro studies, no ethical approval was required.

This review was registered on PROSPERO (registration number: CRD42022362349).

Table 1. Brief report of main data extracted from included papers. Abbreviations: human dysplastic oral keratinocytes (DOK); dysplastic leukoplakic tongue cells (MSK-Leuk1); plaque-induced gingivitis (PG); polymerase chain reaction (PCR); next generation sequencing (NGS).

Study	Population	Intervention (Light Source)	Intervention (PS)	Comparison	Methodology	Outcome
Garg et al. (2012) [24]	DOK	Tungsten filament lamp (500–550 nm; 400 W; 22.7 mW/cm2)	Erythrosine B sodium salt	Control cells incubated with erythrosine B, without PDT exposure	PDT efficacy measurement by quantification of cell viability (one solution cell proliferation assay); cell death morphology analysis; mitochondrial trans-membrane potential analysis; cell death morphology analysis; and caspase activity assay	Erythrosine as an effective photosensitizer for photodynamic therapy (PDT) of oral malignancies
Jin et al. (2022) [25]	DOK	He–Ne ion laser (633 nm, 200 mW/cm2, 10 J/cm2)	Aminolevulinic acid (ALA)	Control were LY2109761, combinate ALA-PDT LY2109761	PDT efficacy measurement by quantification of cell viability (MTT assay); cell apoptosis analysis; wound healing assay; quantitative real-time polymerase chain reaction (RT-qPCR) analysis; Western blot assay; and fluorescence detection	ALA-PDT for suppressing the growth of oral pre-cancerous cells by regulating the transforming growth factor beta (TGF-β) signaling pathway
Le et al. (2022) [26]	MSK-Leuk1	DD4 diode laser (664 nm; 16–38 mW/cm2; 0–10 J/cm2)	Methylene blue (MB)	4 study groups: untreated, MB only, light only, PDT-MB-based; comparisons were CA-9–22 (OSCC cell lines) and human epidermal keratinocytes	PDT efficacy measurement by quantification of cell viability (MTT assay); (RT-qPCR) analysis for matrix metalloproteinases (MMP-9)	Photodynamic therapy with methylene blue for downregulation of matrix metalloproteinases in oral squamous cell carcinoma, oral leukoplakia, and immortalized keratinocytes
Matei et al. (2014) [27]	DOK	He–Ne laser (632.8 nm)	Hydroxy aluminum (AlS2Pc)	Control cells were not incubated with AlS2Pc	PDT effect was assessed with apoptosis analysis, by flow cytometry; protein microarray evaluation (155 apoptosis proteins kit)	photodynamic therapy using aluminum disulfonated phthalocyanine, leading to the induction of apoptosis in dysplastic human oral keratinocytes via the intrinsic mitochondrial apoptotic pathway
Wang et al. (2019) [28]	DOK	Red light source (660 nm; 200 mW/cm2; 6 J/cm2)	Methyl aminolevulinate (MAL)	Comparisons were CA-9–22 (OSCC cell lines) and in vivo Syrian golden hamsters	PDT effect was assessed with apoptosis analysis; terminal deoxynucleotidyl transferase (TdT) assay; flow cytometry; transmission electron microscopy and light microscopy; immunoblotting analysis and ROS detection	MAL-PDT inducing autophagic cell death in DOK oral pre-cancerous cells
Wang et al. (2020) [29]	DOK	He–Ne ion laser (635 nm; 18 mW/cm2; 10 J/cm2)	Aminolevulinic Acid (ALA)	Groups were subdivided by different doses of ALA (0.25, 0.5, 0.75, 1, or 2 mM); comparison was CAL-27 (OSCC cell lines)	PDT effect was assessed with apoptosis analysis, MTT assay and flow cytometry; (RT-qPCR) analysis; Western blot assay and ROS detection	ALA-PDT for killing oral pre-cancerous and oral squamous cell carcinoma cells in a dose- and time-dependent manner in vitro
Wang et al. (2022) [30]	DOK	He–Ne ion laser (635 nm; 108 J/cm2; 200 mW/cm2)	Aminolevulinic acid (ALA) and ALA via chitosan-containing dry hydrogel patch (PACA)	Groups were subdivided by application of ALA-PDT and PACA-PDT; comparison was CAL-27 (OSCC cell lines), in vivo Syrian golden hamsters and in vivo human volunteers	PDT effect was assessed with apoptosis analysis, MTT assay and flow cytometry; ROS detection’ mitochondrial membrane potential (MMP) change detection by fluorescence staining	ALA patch for the in vitro treatment of oral potentially malignant disorders

presents a summary of the results retrieved.

QUIN Tool

Sheth et al. described a useful tool for the risk of bias assessment for in vitro studies in dentistry. The tool is based on the evaluation of the domains: (1) clearly stated aims/objectives; (2) detailed explanation of sample size calculation; (3) detailed explanation of sampling technique; (4) details of comparison group; (5) detailed explanation of methodology; (6) operator details; (7) randomization; (8) method of measurement of outcome; (9) outcome assessor details; (10) blinding; (11) statistical analysis; (12) presentation of results. For each domain, the assessor assigns a value: adequately specified = 2; inadequately specified = 1; not specified = 0. If the question is not applicable in the study, the domain will be excluded from the score calculation. The final score is considered to denote a high risk of bias when QUIN < 50%, a medium risk of bias in ranges from 50% to 70%, and a low risk of bias when QUIN > 70% [31].

3. Results

The preliminary search resulted in 75 studies, out of which 22 were found to be duplicates. After reviewing the titles and abstracts of the remaining 53 studies, 45 were rejected as they did not meet the inclusion criteria (English article; in vitro studies; not review articles; published since 1990). Further evaluation of the full texts led to the exclusion of only one article, since the full text was not available. As a result, seven studies were ultimately identified and included in the analysis. The information regarding these seven selected articles, including details such as the type of photosensitizer, the light source used, the cell lines, and the methodology, are summarized in Table 1. The risk of bias of the studies was assessed with QUIN Tool. Three studies were evaluated as medium-risk (<70% QUIN Tool score), four as low-risk (>70% QUIN Tool score), and noon as serious-risk (<50% QUIN Tool score). The extrapolated data had some heterogeneity that did not allow for a meta-analysis. All the studies provide sound evidence, and none presented a critical RoB in any domain (

Table 2. QUIN Tool scores of in vitro studies. Scores for studies are achieved according to the following: adequately specified = 2; inadequately specified = 1; not specified = 0.

Study	1	2	3	4	5	6	7	8	9	10	11	12	SCORE	Risk of Bias
Garg et al. [24] (2012)	2	0	2	2	2	0	0	2	2	0	2	2	66.7%	Medium
Jin et al. [25] (2022)	2	2	2	2	2	0	2	2	2	0	2	2	83.3%	Low
Le et al. [26] (2022)	2	0	2	2	2	0	2	2	2	0	2	2	75.0%	Low
Matei et al. [27] (2014)	2	0	2	2	2	0	2	2	2	0	2	2	75.0%	Low
Wang et al. [28] (2019)	2	0	2	2	2	0	2	2	2	0	2	2	75.0%	Low
Wang et al. [29] (2020)	2	0	2	2	2	0	0	2	2	0	2	2	66.7%	Medium
Wang et al. [30] (2022)	2	0	2	2	2	0	0	2	2	0	2	2	66,7%	Medium

). The paper from Garg et al. [24] investigated the effectiveness of erythrosine as a photosensitizer for Photodynamic Therapy of oral malignancies. The study found that erythrosine was taken up by both malignant and pre-malignant oral epithelial cells in a dose-dependent manner. However, the percentage of cell killing observed following PDT differed between the two cell lines, with a maximum of 80% for pre-malignant cell killing achieved compared to 60% killing for malignant cells. The study also found that both cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial transmembrane potential (DYm) studies showed that the malignant cells were far more resistant to the changes in DYm when compared to the pre-malignant cells. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high-dose PDT in pre-malignant cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. Matei et al., in 2014 [27], explored the molecular mechanisms activated by PDT using aluminum disulfonated phthalocyanine in dysplastic human oral keratinocytes. The study found that PDT leads to the induction of apoptosis in tumoral cells via the intrinsic mitochondrial apoptotic pathway. The protein microarray analysis was used to evaluate the possible molecular pathways by which PDT activates apoptosis in dysplastic oral keratinocytes cells. Among the assessed analytes, Bcl-2, P70S6K kinase, Raf-1, and bad proteins represent the apoptosis-related biomolecules that showed expression variations with the greatest amplitude. In 2019, the work from Wang et al. [28] reported the use of methyl aminolevulinate photodynamic therapy (MAL-PDT) as a prevention tool against the progression of pre-cancerous lesions to oral cancer. The study found that MAL-PDT induced autophagic cell death in DOK oral pre-cancerous cells and decreased tumor growth in hamster buccal pouch tumors induced by DMBA. The expression of autophagy marker p62/SQSTM1 was increased in DOK cells at 4 h after MAL-PDT treatment, but it was decreased in the pre-cancerous lesions of hamster buccal pouch at 2 weeks after MAL-PDT treatment, suggesting a time-dependent change in the expression of autophagy markers after MAL-PDT treatment. The effects of in vitro PDT on human oral pre-cancerous cell line (DOK) and an oral squamous cell carcinoma cell line (CAL-27) were also evaluated by Wang et al. in 2020 [29] using 5-aminolevulinic acid-mediated photodynamic therapy. The study reported that ALA-PDT inhibited the proliferation of cell lines in a dose- and time-dependent manner. However, the susceptibilities of these cell lines to ALA-PDT were different. The rates of apoptosis were higher in CAL-27 cells than in DOK cells. The study also revealed changes in the expression of matrix metallopeptidase 2 (MMP-2) and MMP-9 after the treatment. Overall, the study suggested that ALA-PDT may be an effective treatment for oral cancer and pre-cancerous lesions. Jin et al. [25] explored the same methodology (ALA-PDT) for the treatment of oral pre-cancerous cells. The study found that ALA-PDT can suppress the growth of oral pre-cancerous cells by regulating the transforming growth factor beta (TGF-β) signaling pathway. The study also found that the suppressive effect of ALA-PDT was enhanced when combined with the TGF-β receptor inhibitor LY2109761. An interesting paper by Le et al. [26] explored the potential of methylene blue (MB) as a photosensitizer for PDT in cell cultures of oral squamous cell carcinoma, oral leukoplakia, and immortalized keratinocytes. The study showed that MB-PDT could significantly decrease the expression of matrix metalloproteinases (MMPs) in all involved cell lines; the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to confirm the efficacy of MB-PDT. Finally, in 2022, Wang et al. [30] analyzed the development of a hydrogel patch called PACA that can be used to deliver ALA for the PDT of oral potentially malignant cells. The study found that PACA hydrogel-mediated PDT (PACA-PDT) improved OPMDs in vitro and in a hamster oral carcinogenesis model. The paper also reported the results of a trial involving 60 OPMD volunteers, which demonstrated the feasibility and comfort of the PACA hydrogel patch.

4. Discussion

OED grading is one of the most valuable prognostic indicators for predicting the transformation of potentially malignant oral cavity disorders to squamous cell carcinoma. It is also used as a rationale for determining management options. Molecular and genetic markers have been identified with encouraging results in determining which potentially malignant disorders are at risk of malignant transformation [3]. Nevertheless, oral cancer is a long-term process; thus, it is possible to design preventive steps to avoid cancerization long before the beginning of malignancy [32]. Conventional therapies for OPDM extend from careful observation to complete resection. Surgery is currently advised for the excision of high-risk lesions; nonetheless, proof of its success is lacking. In some cases, there is an increase in the recurrence of malignancy after surgical excision has been registered; the surgical procedure does not prevent all pre-malignant lesions from evolving into malignancy [33]. Non-surgical therapies should be the approach for lesions affecting an extensive area of the oral mucosa or occurring in patients with high surgical risks or when patients refuse surgical treatment [32]. The treatment of these lesions has always been a matter of debate as early diagnosis and subsequent early application of therapy are still critical in the prognosis and survival of patients with neoplastic oral cavity lesions. Intervening in pre-malignant lesions with a method that is non-invasive, well-tolerated by the patient, and free of side effects on neighboring healthy cells would be an important goal to achieve in improving the therapeutic approach to these lesions. In this context, analyzing the recent literature is a necessary step toward achieving this goal. The study of PDT in vitro is essential in order to fully understand and obtain information on the most effective therapeutic protocols for using this method in treating the pre-malignant lesions of the oral cavity. The seven selected studies analyzed the effects of PDT on dysplastic human cell cultures: six studies on human dysplastic oral keratinocytes (DOK) [24,25,26,27,28,29,30] and only one study on dysplastic leukoplakic tongue cells (MSK-leuk1) [34]. Homogeneity was also evident concerning the light source used as a photo-activating source. All the devices explored were set to similar wavelengths (Table 1). In contrast, considerable heterogeneity was found in photosensitizers. Three of the seven papers used ALA. [25,29,30]. One study applied erythrosine B sodium salt [24], one paper Methylene blue [26], one work hydroxy aluminum [27], and one study methyl aminolevulinate [29]. Although this heterogeneity makes direct comparison of results difficult, it is nevertheless possible to point out that all authors report favorable results. One of the fundamental objectives that in vitro studies can help achieve is establishing the optimal dosage based on the photosensitizer used. Indeed, several authors [24,27] have pointed out that the effects on DOK cells are dose-related. Wang et al. [29] suggested that the optimum inhibition efficiencies for DOK cell lines are 1 mM ALA. This dosage is influenced by the type of photosensitizer and the cell line. Interestingly, while the authors of the study mentioned above showed a better apoptosis-inducing effect in oral cancer CAL-27 cells than in DOK cells, other authors showed a resistance of other oral cancer cell lines (SCC9) to ALA-PDT [35].

Furthermore, the role of matrix metalloproteinases (MMPs) has been indicated in relation to the efficacy of photodynamic therapy in OSCC [36]. MMPs are enzymes that potentiate tumor invasion and metastasis by degrading the extracellular matrix. MMP-2 and MMP-9 are overexpressed in oral dysplasias and oral cancers [37,38,39]. Sharwani et al. [36] registered that after exposure to PDT, the total activity of MMP-2 and MMP-9 in malignant human keratinocytes culture was down-regulated. Authors conclude that PDT causes the suppression of factors responsible for tumor invasion. Similar data are also found in the application of PDT on DOK. Wang et al. 2020, Jin et al. 2022, and Wang et al. 2022 [25,29,30] also observed that the protein levels of MMP-2 and MMP-9 decreased in DOK cell lines treated with 1 mM ALA-induced PDT at 12 h after laser irradiation [29]. Le et al. examined MMPs as markers for the impact of MB PDT and determined that PDT decreased MMP-9 gene expression in cancerous and pre-cancerous cells. MMP-9 could function as valuable markers for evaluating the effects of MB PDT in the therapy of oral cancers and precancers.

5. Conclusions

In conclusion, the data from the studies analyzed showed that (i) erythrosine is an effective photosensitizer for photodynamic therapy (PDT); (ii) ALA-PDT suppresses the growth of oral pre-cancerous cells by regulating the transforming growth factor beta (TGF-β) signaling pathway; ALA-PDT kills oral pre-cancerous cells and oral squamous cell carcinoma cells in vitro in a dose- and time-dependent manner; ALA-PDT is effective for the in vitro treatment of potentially malignant oral disorders; (iii) photodynamic therapy with methylene blue reduced MMPs in oral squamous cell carcinoma, oral leukoplakia, and immortalized keratinocytes; (iv) photodynamic therapy with hydroxy aluminum led to the induction of apoptosis in dysplastic human oral keratinocytes via the intrinsic mitochondrial apoptotic pathway; and (v) MAL-PDT induces autophagic cell death in DOK oral pre-cancerous cells.

Taken together, the data reported by the studies reviewed are promising and confirm the usefulness and efficacy of PDT in treating pre-cancerous cells. These results obtained in vitro lay the foundations for more in-depth in vivo studies that may, in the future, endorse PDT as the technique of choice in the treatment of OPMD.