Photodynamic Priming Improves the Anti-Migratory Activity of Prostaglandin E Receptor 4 Antagonist in Cancer Cells In Vitro

Abstract

The combination of photodynamic agents and biological inhibitors is rapidly gaining attention for its promise and approval in treating advanced cancer. The activity of photodynamic treatment is mainly governed by the formation of reactive oxygen species upon light activation of photosensitizers. Exposure to reactive oxygen species above a threshold dose can induce cellular damage and cancer cell death, while the surviving cancer cells are "photodynamically primed", or sensitized, to respond better to other drugs and biological treatments. Here, we report a new combination regimen of photodynamic priming (PDP) and prostaglandin E₂ receptor 4 (EP4) inhibition that reduces the migration and invasion of two human ovarian cancer cell lines (OVCAR-5 and CAOV3) in vitro. PDP is achieved by red light activation of the FDA-approved photosensitizer, benzoporphyrin derivative (BPD), or a chemical conjugate composed of the BPD linked to cetuximab, an anti-epithelial growth factor receptor (EGFR) antibody. Immunoblotting data identify co-inhibition of EGFR, cAMP-response element binding protein (CREB), and extracellular signal-regulated kinase 1/2 (ERK1/2) as key in the signaling cascades modulated by the combination of EGFR-targeted PDP and EP4 inhibition. This study provides valuable insights into the development of a molecular-targeted photochemical strategy to improve the anti-metastatic effects of EP4 receptor antagonists.

Keywords: antibody-drug conjugate; ovarian cancer; photodynamic therapy; photoimmunotherapy; prostaglandin inhibitor.

Figure 1

Anti-migratory effects of BPD-based PDP, EP4 inhibitor (AH23848), and their combination were evaluated in (A) a gap closure assay using OVCAR-5 cells and (B) the transwell invasion assays using CAOV3 cells. All data are normalized to the vehicle (DMSO) control, and statistical analysis was performed using a one-way ANOVA and post hoc Tukey’s test. Error bars represent the standard error of the mean. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: nonsignificant.

Figure 2

Western blot analysis of p-CREB, CREB, p-EGFR, EGFR, p-ERK1/2, ERK1, ERK2, EP4, and MRP4 in OVCAR-5 cells. Cells were treated with the indicated agents for 24 h, then light-activated (0.1 J/cm², 10 mW/cm²) or maintained in dark conditions. After 24 h, cells were agonized with EGF (50 ng/mL) and PGE2 (1 µM) for 10 min, then whole extracts were collected and analyzed using Western blot. (A) Representative Western blot images and (B–K) relative densitometric bar graphs of phosphorylated and total proteins were shown. Results are normalized to the vehicle control group. Statistical analysis was performed using a one-way ANOVA and post hoc Tukey’s test. Percentages below each band represent the average change in intensity relative to the vehicle control across all experiments. For pERK1 and pERK2 bands, the first number corresponds to pERK1, and the second number corresponds to pERK2. Error bars represent the standard error of the mean. * p ≤ 0.05; ns: nonsignificant. Original western blot images (Supplementary Figure S4).

Figure 3

Conjugation of BPD to cetuximab impacts uptake and gap closure effects in ovarian cancer cells. OVCAR-5 cells were plated in 96-well plates, treated with the indicated BPD or Cet-BPD doses for 24 h, then (A) agents were extracted from cells to quantify cellular photosensitizer uptake, or (B) cells were light-activated at 690 nm and scratched for gap-closure analysis. Representative gap closure images are included (C). Statistical analysis was performed using a one-way ANOVA and post hoc Tukey’s test. Error bars represent the standard error of the mean. ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: nonsignificant.

Figure 4

Investigation of anti-metastatic effects of Cet-BPD-based PDP combined with EP4 inhibition (AH23848). Treatments are evaluated in gap closure assays using OVCAR-5 cells (A) and transwell invasion assays using CAOV3 cells (B). All data are normalized to the vehicle (DMSO) control, and statistical analysis was performed using a one-way ANOVA and post hoc Tukey’s test. Error bars represent the standard error of the mean. ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: nonsignificant.

Figure 5

Western blot analysis of p-CREB, CREB, p-EGFR, EGFR, p-ERK1/2, ERK1, ERK2, EP4, and MRP4 in OVCAR-5 cells. Cells were treated with the indicated agents for 24 h, then light-activated (0.1 J/cm², 10 mW/cm²) or maintained in dark conditions. After 24 h, cells were agonized with EGF (50 ng/mL) and PGE₂ (1 µM) for 10 min, then whole extracts were collected and analyzed using Western blot. (A) Representative Western blot images and (B–K) relative densitometric bar graphs of phosphorylated and total proteins were shown. Results are normalized to the vehicle control group. Statistical analysis was performed using a one-way ANOVA and post hoc Tukey’s test. Percentages below each band represent the average change in intensity relative to the vehicle control across all experiments. For pERK1 and pERK2 bands, the first number corresponds to pERK1, and the second number corresponds to pERK2. Error bars represent the standard error of the mean. * p ≤ 0.05; ** p ≤ 0.01; ns: nonsignificant. Original western blot images (Supplementary Figure S5).

Figure 6

Proposed relationship between the combination treatment (Cet-BPD-based PDP and AH23848) and EGFR-EP4 signal transduction pathways. Arachidonic acid is converted to PGE₂ by COX-1, COX-2, and PGE synthase [38]. PGE₂ is exported from the cell via multiple drug resistance-associated protein 4 (MRP4), where it can bind to the G-protein coupled receptors, EP1–4 [39]. EP4 is coupled to the G protein alpha stimulator (Gs), which activates adenylyl cyclase. Adenylyl cyclase converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), which subsequently activates Protein Kinase A (PKA). When PKA is activated, its catalytic subunits translocate into the nucleus and activate CREB, a transcription factor with complex roles in cancer [40]. EGFR can be activated extracellularly via EGF binding and intracellularly via the EP4/β-arrestin (β-arr)/Src complex [18]. Activated EGFR causes a variety of downstream effects including ERK phosphorylation, which is linked to CREB activation. EP4 has also been shown to induce ERK activation independently of EGFR [41]. The Cet-BPD and EP4 inhibitor combination regimen is designed to simultaneously abrogate EGFR and EP4 signaling to block tumorigenic crosstalk along with overlapping signaling pathways. Abbreviations: AA (arachidonic acid); COX2 (cyclooxygenase-2); PGE2 (prostaglandin E2); MRP4 (multidrug resistance-associated protein 4); EP4 (prostaglandin E2 receptor 4); ATP (adenosine triphosphate); cAMP (cyclic adenosine monophosphate); PKA (protein kinase A); CREB (cyclic AMP response element-binding protein); ERK1/2 (extracellular signal-regulated kinases 1/2); β-arr (β-Arrestin); EGFR (epidermal growth factor receptor); EGF (epidermal growth factor); BPD (benzoporphyrin derivative); Cet (cetuximab).