Alterations in dysadherin expression and F-actin reorganization: a possible mechanism of hypericin-mediated photodynamic therapy in colon adenocarcinoma cells

Abstract

Dysadherin is a recently found anti-adhesion molecule, therefore detection and down regulation of its expression is promising in cancer treatment. The up-regulation of dysadherin contributes to colon cancer recurrence and metastasis. Dysadherin also has connections with cytoskeletal proteins and it can cause alterations in the organisation of filamentous actin (Factin) in metastatic cancers. In this study, hypericin (HYP)-mediated photodynamic therapy (PDT) was performed in two different grade colon adenocarcinoma cell lines HT-29 (Grade I) and Caco-2 (Grade II). Cells were treated with 0.04, 0.08 or 0.15 lM HYP concentrations and irradiated with (4 J/cm 2 ) fluorescent lamps. The effects of HYP was examined 16 and 24 h after the activation. We investigated for the first time the effect of HYP-mediated PDT on the expression of dysadherin and F-actin organisation. According to the results, HYP mediated PDT caused a decrease in gene expression and immunofluorescence staining of dysadherin and an increase in actin stress fibers and actin aggregates in HT-29 and Caco-2 cell lines. Besides, cytotoxicity, number of floating cells and apoptotic index changed depending on the cell type, HYP concentration and incubation time. We have demonstrated for the first time that dysadherin and F-actin could be target molecules for HYP-

Comparison of phototoxic effects of hypericin-mediated photodynamictherapy in HT-29 and Caco-2 colon cancer cells

“…The fluorescent stainings are also presented in Figures 4-7. The induction of apoptosis after PDT was also supported by DAPI staining (Kılıç Süloğlu et al, 2015) and RT-PCR (caspase-3, survivin, Bcl-2, and Bax expressions) (Kılıç Süloğlu et al, 2016).…”

“…Passages 5-15 for HT-29 cells and 10-20 for Caco-2 cells were used for analysis. HYP (HPLC grade, AppliChem, Germany) was prepared as a stock solution in DMSO (final concentration less than 0.1%) and diluted to particular working concentrations according to preliminary experiments (Kılıç Süloğlu et al, 2015). The cells were incubated with different concentrations of HYP (0.04, 0.08, and 0.15 µM) in the dark for 24 h. Prior to irradiation, the medium containing hypericin was replaced with fresh hypericin-free DMEM without phenol red.…”

Evaluation of apoptotic cell death mechanisms induced by hypericin-mediated photodynamic therapy in colon cancer cells

“…According to Kılıç Süloğlu et al (2015), 0.04, 0.08, and 0.15 µM HYP induced cytotoxicity in HT-29 and Caco-2 cells. In this study, the decrease in protein content depending on HYP concentration was parallel to increased cytotoxicity.…”

“…These concentrations caused 20%-42% inhibition of metabolic activity and induced a significant increase in apoptotic index in HT-29 and Caco-2 cells. (Kılıç Süloğlu et al, 2015). In this study, further analysis was performed in order to explain apoptotic mechanisms and related protein alterations due to HYP photoactivation.…”

“…HYP HPLC grade (AppliChem, Germany) was prepared as a stock solution in dimethyl sulfoxide (DMSO) (final concentration, <0.1%). HYP concentrations were chosen according to results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity test (Kılıç Süloğlu et al, 2015). The cells were incubated with 0.04, 0.08, and 0.15 µM HYP concentrations in dark conditions for 24 h. Prior to irradiation, the medium containing HYP was replaced with fresh HYP-free DMEM.…”