O2 and Ca(2+) fluxes as indicators of apoptosis induced by rose bengal-mediated photodynamic therapy in human oral squamous carcinoma cells

Photomed Laser Surg. 2015 May;33(5):258-65. doi: 10.1089/pho.2014.3863.

Abstract

Objective: Photodynamic therapy (PDT) triggers various cellular responses and induces cell death via necrosis and/or apoptosis. This study evaluated the feasibility of using O2 and Ca(2+) fluxes as indicators of apoptosis induced by rose bengal (RB)-mediated PDT in human oral squamous carcinoma cells (Cal27 cells).

Methods: Intracellular reactive oxygen species (ROS) generation was assessed by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. Real-time O2 and Ca(2+) flux measurements were performed using the noninvasive micro-test technique (NMT). Apoptosis of the PDT-treated cells was confirmed by 4'6-diamidino-2-phenylindole-dilactate staining. The activation of apoptosis-related molecules was examined using Western blot. We assayed the effects of the fluctuation of O2 and Ca(2+) flux in response to PDT and the apoptotic mechanism, by which ROS, O2, and Ca(2+) synergistically may trigger apoptosis in PDT-treated cells.

Results: Real-time O2 and Ca(2+) flux measurements revealed that these indicators were involved in the timely regulation of apoptosis in the PDT-treated cells and were activated 2 h after PDT treatment. RB-mediated PDT significantly elicited the generation of ROS by approximately threefold, which was critical for PDT-induced apoptosis. Cytochrome c and cleaved caspase-3, caspase-9 and poly ADP ribose polymerase (PARP) were overexpressed, and the data provided evidence that 2 h was considered to be the key observation time in RB-mediated PDT-induced apoptosis in Cal27 cells.

Conclusions: Our collective results indicated that the effects of O2 and Ca(2+) fluxes may act as a real-time biomonitoring system of apoptosis in the RB-PDT-treated cells. Also, RB-mediated PDT can be a potential and effective therapeutic modality in oral squamous cell carcinoma.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / radiation effects*
  • Carcinoma, Squamous Cell / metabolism*
  • Carcinoma, Squamous Cell / pathology
  • Carcinoma, Squamous Cell / radiotherapy
  • Cell Culture Techniques
  • Cell Line, Tumor
  • Fluorescent Dyes / pharmacology*
  • Humans
  • Mouth Neoplasms / metabolism*
  • Mouth Neoplasms / pathology
  • Mouth Neoplasms / radiotherapy
  • Reactive Oxygen Species / metabolism*
  • Rose Bengal / pharmacology*

Substances

  • Fluorescent Dyes
  • Reactive Oxygen Species
  • Rose Bengal