Autophagy upregulation may explain inhibition of oral carcinoma in situ by photobiomodulation in vitro

https://doi.org/10.1016/j.jphotobiol.2021.112245Get rights and content

Highlights

  • Photobiomodulation triggers autophagy markers in oral carcinoma cells.

  • Upregulation of autophagy markers inhibit cell proliferation in vitro.

  • Co-cultures of malignant and stromal cells behave differently to isolated cells.

  • Manipulation of autophagy could be a strategy to treat oral carcinoma in vivo.

Abstract

There is currently no clear understanding on the pathways involved in the process of cell inhibition by photobiomodulation (PBM). The present study evaluated the influence of PBM on the expression of autophagy markers in vitro in an in situ model of oral carcinoma. Oral squamous cell carcinoma (Cal27) and stromal fibroblasts (FG) cultures were used. The independent variables were ‘cell type’ (FG and CAL27) ‘culture condition’ (monocultures or co-cultures) and PBM (placebo and 36 J/cm2). The cultures were irradiated from a red LED source for mRNA expression and protein expression analyses. The autophagy markers evaluated were Beclin-1, LC3B and p62 as well as adjuvant markers (BAX Bcl-2, VEGF, CD105, CD34, PRDX1, PRDX4 and GRP78). The Cal27 cells upregulated the autophagy markers upon exposure to PBM both at the mRNA and protein expression levels, providing evidence to explain malignant cell inhibition by PBM.

Introduction

Oral squamous cell carcinoma (OSCC) is the commonest malignant neoplasm affecting the oral cavity [1] and despite advances in Science, early diagnosis and treatment remain the most important factors on the outcome, particularly quality of life [2,3,4]. Most such lesions may develop from potentially malignant oral diseases (OPMD), which consist of asymptomatic lesions detected in the oral mucosa upon clinical examination [2,4,5,6,7]. Despite several proposals for management of carcinogenesis, satisfactory protocols are yet to be defined and, in the meantime, the gold standard protocol is biopsy, histopathological identification of oral dysplasia and clinical follow-up [6,7]. The end stage in the process of carcinogenesis based on the evolution of epithelial dysplasia prior to frank invasion is carcinoma in situ (CIS) [8]. Intercepting such lesions prior to invasion could have a potential positive impact on treatment outcome.

A previous study by Takemoto et al. [9] used high energy densities (36 J/cm2) based on red LED irradiation and demonstrated an inhibitory effect on the formation of malignant cell colonies in co-culture with stromal fibroblasts after 72 h of daily stimulation. It is important to therefore identify and understand those mechanisms involved in the process of carcinogenesis that are affected by PBM. Once this is achieved, manipulating such processes may optimize traditional treatment strategies [10,11,12].

Autophagy is one the main mechanisms involved in carcinogenesis, regulating cell survival in critical situations, such as starvation and hypoxia, and when overactivated can lead to upregulation of apoptotic signals and consequently cell death [[39], [47], [66],16]. This mechanism has recently been investigated under the influence of high-dose PBM (20 J, 685 nm, 50 mW) in cervical adenocarcinoma (HeLa) cell cultures, demonstrating promising optimization of radiosensitivity in inducing apoptosis associated with autophagy [12].

The aim of this study was to evaluate the influence of PBM on autophagy markers in a CIS model in vitro. The null hypothesis tested was that PBM has no effect on autophagy markers in CIS.

Section snippets

Materials and Methods

This study was approved by the Research Ethics Committee of Faculdade São Leopoldo Mandic, Brasil, protocol number 2,604,280.

mRNA Expression of Autophagy and Apoptosis-Related Genes

LC3B expression was not affected by PBM in any of the culture conditions (P = 0.187). Fibroblasts down regulated LC3B when in co-culture with Cal27 (P < 0.0001). Conversely, the culture conditions did not influence BECN1 expression (P = 0.874). PBM upregulated BECN1 in CAL27 cultures both in the monocultures (P < 0.0001) and in the co-cultures (P = 0.0029) (Fig. 1).

Similar to LC3B, BAX expression was not affected by PBM in the fibroblast cultures (P = 0.594), though the co-cultures

Discussion

Although the use of cell culture is considered an important tool for understanding cell biology in carcinogenesis, the use of isolated cell lines lacks the interaction between malignant cells and stromal cells ([17,[35], [67]]). Co-culture models therefore facilitate the study of carcinogenesis and treatment strategies based on tumor microenvironment observations [18]. The present study adopted a co-culture model separated by a semipermeable membrane in an attempt to mimic the cellular

Conclusions

Oral squamous cell carcinoma cells exposed to PBM significantly increased BECN1, which was accompanied by an increase in Beclin-1, LC3B and p62 at the protein level. Beclin-1, Bcl2 and BAX expression point to malignant cell inhibition rather than cell death outcome in response to PBM. The co-culture model showed an important difference in autophagy markers both in gene expression and protein synthesis when compared to monocultures.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the São Paulo Research Foundation (FAPESP), grant number 2017/06579-1.

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