Synergistic Cytotoxicity of a Toxin Targeting the Epidermal Growth Factor Receptor and the Glycosylated Triterpenoid SO1861 in Prostate Cancer

Abstract

Treatment of advanced prostate cancer lacks specificity and curative intent. Therefore, the need for new targeted therapeutic approaches is high. In the present study, we generated the new targeted toxin EGF-PE24mutΔREDLK binding to the epidermal growth factor receptor (EGFR) on the surface of prostate cancer cells. It consists of the human epidermal growth factor (EGF) as binding domain and a de-immunized variant of Pseudomonas Exotoxin A (PE), called PE24mutΔREDLK, as toxin domain. The toxin domain contains a deletion of the C-terminal KDEL-like motif REDLK to prevent its transport from sorting endosomes via the KDEL receptor mediated pathway into the cytosol, where it can inhibit cellular protein biosynthesis and induce apoptosis. Indeed, REDLK deletion resulted in a strong decrease in cytotoxicity of the targeted toxin in prostate cancer cells compared to the parental targeted toxin EGF-PE24mut. However, addition of the plant glycosylated triterpenoid SO1861, which is known to mediate the release of biomolecules from endolysosomal compartments into the cytosol, resulted in an up to almost 7,000-fold enhanced synergistic cytotoxicity. Moreover, combination of PE24mutΔREDLK with SO1861 led to a cytotoxicity that was even 16- to 300-fold enhanced compared to that of EGF-PE24mut. Endolysosomal entrapment of the non-toxic targeted toxin EGF-PE24mutΔREDLK followed by activation through enhanced endosomal escape therefore represents a new promising approach for the future treatment of advanced prostate cancer with high efficacy and diminished side effects.

Keywords: EGF; EGFR; SO1861; endosomal escape enhancer; prostate cancer; targeted toxin.

Figure 1

Generation of the targeted toxin EGF-PE24mutΔREDLK. (A) Schematic representation of the targeted toxin in the expression vector pHOG21. (B) SDS-PAGE and Western Blot of EGF-PE24mutΔREDLK. The targeted toxin was found in the elution fractions EF3 and EF4 after purification by IMAC (arrow). Abbreviations: PE, periplasmatic extract; FT, flow through; EF, elution fraction, M, protein marker.

Figure 2

Cytotoxicity of EGF-PE24mutΔREDLK in combination with SO1861 in prostate cancer cells. (A) Cell viability was determined by WST-1 assay after 48 h (LNCaP) or 72 h incubation (DU145, PC-3, CHO) with EGF-PE24mutΔREDLK alone or in combination with a subtoxic concentration of 1 µg/ml SO1861. Mean values +/- SEM of three independent experiments. Statistical significance was calculated by unpaired t-test with Welch's correction with ns, not significant; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Inhibition of protein biosynthesis and induction of apoptosis in prostate cancer cells after treatment with EGF-PE24mutΔREDLK and SO1861. (A) Protein biosynthesis inhibition was demonstrated by puromycin Western Blot after 48 h in LNCaP and after 72 h in DU145 and PC-3 cells. EGFR-negative CHO cells remained unaffected. (B) Induction of apoptosis after combination treatment was marked by poly (ADP-ribose) polymerase (PARP) cleavage and caspase-3 activation. Western blots after 48 or 72 h of single or combination treatment of LNCaP (48 h), DU145, PC-3 or CHO cells (all 72 h) with 1.0 µg/ml SO1861 and 2.5 nM EGF-PE24mutΔREDLK. β-actin was used as a loading control. * As CHO cells are not of human origin, detection of human Caspase-3 was not possible in this cell line. Abbreviation: M, protein marker.

Figure 4

Induction of apoptosis and necrosis in prostate cancer cells after treatment with EGF-PE24mutΔREDLK and SO1861. (A) Cell morphology in phase-contrast microscopy at 20× magnification after 48 h or 72 h single or combination treatment with 2.5 nM EGF-PE24mutΔREDLK plus 1.0 µg/ml SO1861. Morphological changes during apoptosis were cell rounding, shrinking, blebbing (dotted arrow) and formation of apoptotic bodies (arrows). (B) Cells were stained with AV (FL1-H) and EthD-III (FLH-2) and analyzed by flow cytometry to distinguish between living cells (AV^-/ EthD-III^-), early apoptosis (AV⁺/ EthD-III^-), late apoptosis (AV⁺/ EthD-III⁺) and necrosis (AV^-/ EthD-III⁺).