Pancreatic cancer-associated Cathepsin E as a drug activator

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is challenging to treat, and better means to detect and/or treat pancreatic cancer are urgently needed to save lives. Cathepsin E (Cath E) is a proteolytic enzyme highly expressed in PDAC. In this study, a novel approach using Cath E activation of a Cath E-specific prodrug was demonstrated. Specific activation of the prodrug is expected to kill pancreatic cancer cells without harming normal pancreatic cells. A novel 5-aminolevulinic acid (5-ALA) prodrug was custom-designed to be activated selectively by endogenous Cath E within the PDAC cells. The 5-ALA prodrug was incubated with Cath E-positive and -negative tumor cells and illuminated with various doses of light. In addition, mice genetically engineered to develop PDAC were injected intravenously with the 5-ALA prodrug, and the pancreas was treated with light irradiation. One day after treatment, PDAC tissue was assessed for apoptosis. The 5-ALA prodrug was activated within the Cath E-positive tumor but not in the normal pancreatic tissue. When used in combination with light treatment, it allowed delivery of selective photodynamic therapy (PDT) to the cancerous tissues, with minimal harm to the adjacent normal tissues. With this novel Cath E activation approach, it is possible to detect pancreatic cancer cells accurately and specifically impair their viability, while sparing normal cells. This treatment could result in fewer side effects than the non-specific treatments currently in use. Cath E is a specific and effective drug activator for PDAC treatment.

Figure 1. 5-ALA prodrug is efficiently activated by Cath E-expressing PDAC cells

(A) Representative microscopic images of unfixed human pancreatic cancer cells (MPanc96-CTSE) after treatment with 0.5 μM 5-ALA prodrug (a, and b) free 5-ALA as a positive control (c, d). Cells imaged on an inverted epifluorescence microscope in bright field (top) and TRITC (Tetramethyl Rhodamine isothiocyanate) channel (λ_ex=557 nm, λ_em=576 nm) (bottom). Images show significant fluorescence signals originating from cells treated with the 5-ALA prodrug, comparable to that obtained with free 5-ALA, indicating the efficient release of 5-ALA from the prodrug. (B) 5-ALA prodrug activation is Cath E-dependent. Representative microscopic images of unfixed PDAC cells expressing Cath E (MPanc96-CTSE) (a, b) and parent cell line (MPanc96-FG30) (c, d) after treatment with 0.5 μM 5-ALA prodrug for 1hr at 37°C. The pronounced fluorescence signal observed within the MPanc96-CTSE cells indicates strong Cath E-mediated release of 5-ALA, which enables cells visualization in the TRITC channel (b). In contrast, MPanc96-FG30 cells, with limited Cath E expression, failed to show an appreciable fluorescence signal, strongly suggesting the lack of free 5-ALA within the cells (d).

Figure 2. 5-ALA prodrug, in combination with light treatment, is an effective phototoxic agent that selectively and sensitively induces cell death in vitro

Microscopic image of unfixed human pancreatic cancer cells (MPanc96-CTSE) untreated (top) and treated with 0.1 μM 5-ALA prodrug (bottom), before (a, c) and after (b, d) illumination with doses of 18 J/cm² (top) and 10 J/cm² (bottom). The cells show no sign of morphological changes upon exposure to high doses of light alone. However, massive cell destruction is observed upon treatment with the 5-ALA prodrug and exposure to a low dose of light.

Figure 3. 5-ALA prodrug is an effective phototoxic agent in cells in the presence of light

(A) Human pancreatic cancer cells, MPanc96-FG30 (left) and MPanc96-CTSE (right), treated with 0.1 μM 5-ALA prodrug after light exposure (10 J/cm²). The images show morphological changes in MPanc96-CTSE cells and, to a lesser extent, in the parental MPanc96-FG30 cells, which have limited expression of Cath E. This strongly suggests that the enzymatic activity of Cath E plays a major role in the efficiency of treatment. (B) Viability of MPanc-FG30 and MPanc96-CTSE cells treated with light dose of 2.5 J/cm² with various concentrations (0.1, 0.5, 1, and 5 μM) of 5-ALA prodrug. Quantitation of cell viability demonstrated that the PDT was more highly phototoxic in MPanc96-CTSE, compared to Mpanc-FG30, cells under all conditions. (C) Viability of MPanc96-CTSE cells treated with a range of light doses (2.5, 5, 10, and 15 J/cm²) at various concentrations (0.1, 0.5, 1, and 5 μM) of 5-ALA prodrug. Quantitation of cell viability illustrated that the phototoxic effect of PDT on the cells increased with increasing concentration of 5-ALA prodrug and light dose.

Figure 3. 5-ALA prodrug is an effective phototoxic agent in cells in the presence of light

(A) Human pancreatic cancer cells, MPanc96-FG30 (left) and MPanc96-CTSE (right), treated with 0.1 μM 5-ALA prodrug after light exposure (10 J/cm²). The images show morphological changes in MPanc96-CTSE cells and, to a lesser extent, in the parental MPanc96-FG30 cells, which have limited expression of Cath E. This strongly suggests that the enzymatic activity of Cath E plays a major role in the efficiency of treatment. (B) Viability of MPanc-FG30 and MPanc96-CTSE cells treated with light dose of 2.5 J/cm² with various concentrations (0.1, 0.5, 1, and 5 μM) of 5-ALA prodrug. Quantitation of cell viability demonstrated that the PDT was more highly phototoxic in MPanc96-CTSE, compared to Mpanc-FG30, cells under all conditions. (C) Viability of MPanc96-CTSE cells treated with a range of light doses (2.5, 5, 10, and 15 J/cm²) at various concentrations (0.1, 0.5, 1, and 5 μM) of 5-ALA prodrug. Quantitation of cell viability illustrated that the phototoxic effect of PDT on the cells increased with increasing concentration of 5-ALA prodrug and light dose.

Figure 4. Mechanism of 5-ALA prodrug-induced cell death

Representative microscopic images of untreated (top) and treated (bottom) MPanc96-CTSE cells, illustrating apoptotic and necrotic cell damage that occurred during PDT. The images indicate that apoptosis is the primary mechanism of cell death. Cells were stained with both Yo-Pro (apoptosis) and PI (necrosis) immediately after PDT.

Figure 5. 5-ALA prodrug in combination with light treatment caused selective pancreatic cancer cell death in vivo

(a–c) Normal pancreas and (d–f) mouse PDAC from genetic mouse model (p53 conditional deletion/LSL-Kras^G12D/Pdx1-Cre) treated with 10 J/cm². (a, d) negative control saline, (b, e) positive control free 5-ALA, and (c, f) 5-ALA prodrug (1 mg 5-ALA equivalent/kg). Formalin-fixed, paraffin-embedded tissue sections were examined for apoptosis by TUNEL using ApopTag Peroxidase Kit. TUNEL staining of PDAC pancreas showed no apoptotic cells in mice treated with saline and PDT (d). However, multiple brown-stained cancer cells were observed in pancreas of animals treated with free 5-ALA and 5-ALA prodrug, indicating apoptosis (e and f, arrows). Tissue sections of normal pancreas of mice treated with PDT using 5-ALA prodrug showed no apoptotic staining, 5-ALA prodrug similar to the negative saline control (a, c). In contrast, scattered brown-stained spots were observed in normal acinar cells in the tissue sections of normal pancreas of mice treated with PDT using free 5-ALA (b, arrows).